Methods for quantifying inter-alpha inhibitor proteins

ABSTRACT

Described herein are methods for quantifying lAIP levels in a sample (e.g., from a subject) using lAIP ligand-based assays. Also disclosed are methods for measuring lAIP-IAIP ligand complexes, and methods of evaluating, monitoring, and treating subjects using the aforementioned lAIP quantification methods.

BACKGROUND

IAIP (Inter alpha Inhibitor Proteins) are a family of naturallyoccurring, immunomodulatory plasma proteins that circulate in highconcentrations in the blood of all mammals. IAIP are primarily producedin the liver, released into the blood and a subunit (bikunin) isexcreted in the urine. IAIP have an important role in modulatinginflammation. They have extensive protective effects toward the seriousinflammation caused by infection, trauma, and injury and importantly,the protective effects of IAIP are independent from the causativemicrobial agents or triggers. Members of this family are composed ofheavy and light polypeptide subunits that are covalently linked byglycosaminoglycan. IAIP can be found in vivo as Inter-alpha-Inhibitor(IαI), a 250 kDa molecule composed of two heavy chains (H1 & H2) and asingle light chain (L), and Pre-alpha-Inhibitor (PαI), a 125 kDamolecule composed of one heavy (H3) and one light chain (L).

When the body generates inflammatory signals, such as those elicitedduring injury or infection, IAIP traffic into the tissues and directlyreach sites of inflammation. The heavy chains of IAIP enhance theanti-inflammatory response by binding to proteins which are part of theinflammatory cascade. Also, when the heavy chains are cleaved, the lightchain with its associated GAG (named Bikunin due to its two Kunitzdomains) is released and the serine protease inhibitory activity of thelight chain is activated. Bikunin inhibits the activity of serineproteases such as trypsin, elastase, plasmin, cathepsin G, and furin.IAIP exert their anti-inflammatory effects through multiple mechanisms.They have been shown to bind proteins that amplify inflammation, such ascomplement and extracellular histones (Damage Signals), thus attenuatinginflammatory processes. Through the heavy chains, IAIP can bind theextra cellular matrix (ECM) proteins and have been shown to promote lungepithelial repair after injury in both in vitro and in vivo models. IAIPhave also been shown in multiple in vivo models to down regulateinflammatory cytokines, such as TNF-α and IL-6. Bikunin-deficient (andtherefore IAIP deficient) mice have been shown to have decreasedinflammatory markers of cell adhesion, VCAM-1 and ICAM-1.

In healthy individuals, the amount of circulating IAIP in blood isrelatively high (between 400-800 mg/L). However, IAIP levels rapidlydecrease during systemic inflammation/sepsis in newborns and in adultpatients (Baek Y W, et al. J Pediatr. 2003; 143:11-15; Lim Y P, et al. JInfect Dis. 2003; 188:919-926 and Opal S M, et al. Crit Care Med. 2007;35:387-392), and decreased levels of IAIP have been shown to correlatestrongly with disease progression. As diseases progress to more advancedand life-threatening stages, IAIP levels drop precipitously, suggestingthat IAIP has clinical utility as a prognostic and theranostic marker inassisting clinicians in monitoring disease progression and makinginformed treatment decisions for diseases such as severe inflammatorydiseases such as severe pneumonia, sepsis and the associated organdamage, NEC, wound healing, burn, cancer, stroke, Alzheimer's disease,epilepsy and others.

A standardized competitive IAIP immunoassay has been used to measureIAIP in over blood samples from patients with systemic inflammationfollowing bacterial and viral infections. The competitive IAIPimmunoassay provides a measure of IAIP that detects only the lightchain, therefore both intact IAIP and cleaved bikunin are detected inthis assay. The competitive assay does not detect the other crucialparts of IAIP which are very important for its anti-inflammatory andtissues repair properties; namely the heavy chains and theglycosaminoglycan. Thus, the competitive IAIP immunoassay haslimitations in assessment of active IAIP in a patient sample. Given theimportance of IAIP as a crucial component of the body's protectiveinnate immune defenses and its potential use as a prognostic biomarker,there exists a need for improved methods to quantitatively measure IAIP.An ideal assay would measure both the light chain and heavy chainsubunits to capture the complete molecule.

SUMMARY OF THE INVENTION

The present invention provides methods for quantifying IAIP in a samplefrom a subject through direct detection with agents that bind to anddetect IAIP (e.g., agents that bind to intact IAIP, a heavy chain ofIAIP, or a glycosaminoglycan (GAG) of IAIP). The methods ofquantification can be used to evaluate, diagnose, treat, or monitorsubjects, or to evaluate disease severity or treatment efficacy in asubject. The invention also features kits that can be used to quantifyIAIP according to the methods described herein.

In a first aspect, featured is a method for quantifying inter-alphainhibitor protein (IAIP) in a sample from a subject by: a) contactingthe sample with a binding agent to produce an IAIP-binding agentcomplex, wherein the binding agent is bound to a support; b) contactingthe IAIP-binding agent complex with a detection agent; and c) detectingan amount of the detection agent bound to the IAIP-binding agent complexto quantify IAIP in the sample.

In some embodiments, the IAIP is intact IAIP.

In some embodiments, the binding agent is an IAIP ligand that binds toIAIP. In some embodiments, the binding agent is an antibody thatspecifically binds to IAIP.

In some embodiments, the detection agent is or contains an IAIP ligand.In some embodiments, the detection agent further contains an antibodythat binds to the IAIP ligand detection agent (e.g., an IAIP ligand). Insome embodiments, the detection agent is an antibody that specificallybinds to IAIP. For example, in particular embodiments, the binding agentis an antibody that specifically binds to IAIP (e.g., MAb 69.26 or MAb69.31) and the detection agent is or contains an IAIP ligand that bindsto IAIP (e.g., heparin, hyaluronic acid, endotoxin (LPS), or a histone).In other embodiments, the binding agent is an an IAIP ligand that bindsto IAIP (e.g., heparin, hyaluronic acid, LPS, or a histone) and thedetection agent is an antibody that specifically binds to IAIP (e.g.,MAb 69.26 or MAb 69.31).

In some embodiments, the IAIP is in an IAIP-IAIP ligand complex.

In some embodiments, the binding agent is an IAIP ligand that binds toIAIP. In some embodiments, the IAIP ligand of the IAIP-IAIP ligandcomplex is different from the binding agent. In some embodiments, thebinding agent is an antibody that binds to the IAIP ligand of theIAIP-IAIP ligand complex. In some embodiments, the binding agent is anantibody that specifically binds to IAIP of the IAIP-IAIP ligandcomplex.

In some embodiments, the detection agent contains an IAIP ligand thatbinds to IAIP. In some embodiments, the detection agent further containsan antibody that binds to the IAIP ligand detection agent. In someembodiments, the IAIP ligand of the IAIP-IAIP ligand complex isdifferent from the IAIP ligand detection agent. In some embodiments, thedetection agent is an antibody that binds to the IAIP ligand of theIAIP-IAIP ligand complex. In some embodiments, the detection agent is anantibody that specifically binds to IAIP of the IAIP-IAIP ligandcomplex.

In some embodiments, the antibody is a monoclonal antibody. In someembodiments, the antibody is MAb 69.26 or MAb 69.31.

In some embodiments, the IAIP ligand is selected from the groupconsisting of endotoxin (LPS), heparin, a histone, hyaluronic acid,vitronectin, fibronectin, laminin, tenascin C, aggrecan, von WillebrandFactor, pentraxin-3 (PTX3), TNF-stimulated gene-6 (TSG-6), factor IX, acomplement component, factor XIIIa, and tissue transglutaminase. In someembodiments, the complement component is C1q, C2, C3, C4, C5, C6, C8,properdin, or factor D.

In some embodiments, the detection agent contains a label. In someembodiments, the label is biotin, an enzyme, an enzyme substrate, aradiolabel, a luminescent compound, colloidal gold, a particle, or afluorescent dye.

In some embodiments, the support is a plate, a particle, a nanoparticle,a resin, a membrane, a biochip, a container, a test strip, or a bead.

In some embodiments, the method further includes a wash step betweensteps a) and b).

In some embodiments, the method further includes a wash step betweensteps b) and c).

In some embodiments, the method further includes a blocking step priorto step a) or step b).

In some embodiments, the contacting in step a) and/or b) is performed ata pH of about 7.0 to about 3.5. In some embodiments, the pH is about 5.0to about 3.5. In some embodiments, the pH is about 4.0.

In some embodiments, the sample is a fluid. In some embodiments, thefluid is blood, plasma, serum, urine, cerebrospinal fluid, synovialfluid, amniotic fluid, interstitial fluid, follicular fluid, peritonealfluid, bronchoalveolar lavage fluid, breast milk, sputum, lymph, bile,or tissue homogenate.

In some embodiments, the subject is a human subject. In someembodiments, the subject has been identified as having or at risk ofdeveloping an inflammatory disease or condition or an infection. In someembodiments, the subject has not been identified as having or at risk ofdeveloping an inflammatory disease or condition or an infection. In someembodiments, the method is performed before, after, or concurrent withdiagnosis of the subject as having or at risk of an inflammatory diseaseor condition or an infection. In some embodiments, the method isperformed substantially concurrent with treatment of the subject for aninflammatory disease or condition or an infection. In some embodiments,the method is performed prior to treatment of the subject for aninflammatory disease or condition or an infection. In some embodiments,the method is performed after treatment of the subject for aninflammatory disease or condition or an infection.

In some embodiments, the inflammatory disease or condition is selectedfrom the group consisting of sepsis, septic shock, sterile sepsis,trauma, injury, stroke, acute inflammatory disease, SIRS, acute lunginjury, ARDS, pneumonia, necrotizing enterocolitis, acute pancreatitis,renal disease, acute kidney injury, liver injury, acute circulatoryfailure, preeclampsia, cancer, cancer metastasis, tumor invasion,peripheral artery disease, type 1 or type 2 diabetes, atheroscleroticcardiovascular disease, intermittent claudication, critical limbischemic disease, myocardial infarction, carotid occlusion, umbilicalcord occlusion, low birth-weight, premature birth, surgery-inducedinflammation, abscess-induced inflammation, multiple sclerosis,pulmonary insufficiency, peripheral neuropathy, hypoxic ischemia,bacterial infection, wounds, burns, lacerations, contusions, bonefractures, surgical procedures, tissue ischemia, rheumatoid arthritis,meningitis, inflammatory bowel disease, chronic obstructive pulmonarydisease, rhinitis, preterm labor, or an infectious disease.

In some embodiments, the infection is caused by a gram negativebacteria, such as Neisseria species including Neisseria gonorrhoeae andNeisseria meningitidis, Branhamella species including Branhamellacatarrhalis, Escherichia species including Escherichia coli,Enterobacter species, Proteus species including Proteus mirabilis,Pseudomonas species including Pseudomonas aeruginosa, Pseudomonasmallei, and Pseudomonas pseudomallei, Klebsiella species includingKlebsiella pneumoniae, Salmonella species, Shigella species, Serratiaspecies, Acinetobacter species; Haemophilus species includingHaemophilus influenzae and Haemophilus ducreyi, Brucella species,Yersinia species including Yersinia pestis and Yersinia enterocolitica,Francisella species including Francisella tularensis, Pasteurellaspecies including Pasteurella multocida, Vibrio cholerae, Flavobacteriumspecies, meningosepticum, Campylobacter species including Campylobacterjejuni, Bacteroides species (oral, pharyngeal) including Bacteroidesfragilis, Fusobacterium species including Fusobacterium nucleatum,Calymmatobacterium granulomatis, Streptobacillus species includingStreptobacillus moniliformis, and Legionella species includingLegionella pneumophila.

In some embodiments, the subject is a neonate, a child, an adolescent,or an adult.

In some embodiments, the method is performed one or more times per year.In some embodiments, the method is performed one or more times permonth. In some embodiments, the method is performed one or more timesper week. In some embodiments, the method is performed one or more timesper day. In some embodiments, the method is performed one or more timesper hour.

In some embodiments, the method is performed at least once, at leasttwice, at least three times, at least five times, or at least ten times.

In some embodiments, the method further includes administering atreatment comprising IAIP or a therapeutic agent to the subject. In someembodiments, the subject has an IAIP concentration of 200 μg/mL orlower. In some embodiments, the sample from the subject has an elevatedlevel of IAIP-IAIP ligand complex relative to a reference sample. Insome embodiments, the subject has or is at risk of developing aninflammatory disease or condition or an infection.

In some embodiments, the method includes administering IAIP and atherapeutic agent to the subject.

In some embodiments, the therapeutic agent is selected from the groupconsisting of an antibiotic agent, an antiviral agent, an antifungalagent, an antiparasitic agent, an anti-inflammatory agent, ananti-cancer agent, an anti-coagulant, an immunomodulatory agent, abronchodilator agent, a complement inhibitor, a vasopressor, a sedative,or mechanical ventilation.

In some embodiments, the subject has been ill for at least one day. Insome embodiments, the subject has been ill for at least one week. Insome embodiments, the subject has been ill for at least one month. Insome embodiments, the subject has been ill for at least one year.

In some embodiments, the method is for: a) evaluating the health statusof the subject; b) monitoring the health status of the subject; c)diagnosing the subject as having or being at risk for an inflammatorydisease or condition or an infection; d) evaluating efficacy of atreatment administered to the subject; or e) evaluating disease severityin the subject.

In some embodiments, the method further includes comparing the amount ofIAIP and/or an IAIP-IAIP ligand complex detected in the sample to theamount of IAIP and/or an IAIP-IAIP ligand complex found in a sample froma normal subject or to a cutoff value. In some embodiments, an amount ofIAIP in the sample that is lower than an amount of IAIP in the samplefrom the normal subject or relative to the cutoff value indicates thatthe subject has or is at risk of developing an inflammatory disease orcondition or an infection. In some embodiments, an amount of anIAIP-IAIP ligand complex in the sample that is greater than an amount ofIAIP-IAIP ligand complex in the sample from the normal subject orrelative to the cutoff value indicates that the subject has or is atrisk of developing an inflammatory disease or condition or an infection.In some embodiments, the amount of IAIP in the sample from the normalsubject, or the cutoff value, is >250 μg/mL. In some embodiments, theamount of IAIP in the sample from the normal subject is about 260 toabout 540 μg/mL.

In some embodiments, a determination that the subject has an IAIPconcentration of 250 μg/mL or less indicates that the subject has or isat high risk of developing an inflammatory disease or condition or aninfection or is diagnosed as having an increased risk of morbidityand/or mortality.

In some embodiments, the subject has an IAIP concentration of 200 to 300μg/mL. In other embodiments the method is performed at least once ayear, at least twice a year, at least once a month, at least once aweek, at least once a day, or at least once an hour.

In some embodiments, the subject previously had an inflammatory diseaseor condition or an infection.

In some embodiments, the method is performed prior to the treatmentand/or one or more times during the course of the treatment. In someembodiments, the method is performed after initiation of the treatmentand/or after conclusion of the treatment. In some embodiments, thetreatment is determined to be effective if the concentration of IAIPincreases in the subject relative to a prior measurement of IAIP in thesubject and/or if the concentration of an IAIP-IAIP ligand complexdecreases in the subject relative to a prior measurement of an IAIP-IAIPligand complex in the subject. In some embodiments, the treatment isdetermined to be ineffective if the concentration of IAIP decreases orremains constant in the subject relative to a prior measurement of IAIPin the subject and/or if the concentration of an IAIP-IAIP ligandcomplex increases or remains constant in the subject relative to a priormeasurement of an IAIP-IAIP ligand complex in the subject. In someembodiments, the method further comprises modifying or changing thetreatment.

In a second aspect, featured is a method of treating a subject (e.g., ahuman, such as a neonate, a child, an adolescent, or an adult) that hasor is at risk of developing an inflammatory disease or infection, inwhich the subject has been determined to be in need of treatmentaccording to the method of the first aspect, by administering to thesubject a therapeutically effective amount of IAIP and/or a therapeuticagent selected from the group consisting of an antibiotic agent, anantiviral agent, an antifungal agent, an antiparasitic agent, ananti-inflammatory agent, an anti-cancer agent, an anti-coagulant, animmunomodulatory agent, a bronchodilator agent, a complement inhibitor,a vasopressor, a sedative, or mechanical ventilation.

In some embodiments, the inflammatory disease or condition is selectedfrom the group consisting of sepsis, septic shock, sterile sepsis,trauma, injury, stroke, acute inflammatory disease, SIRS, acute lunginjury, ARDS, pneumonia, necrotizing enterocolitis, acute pancreatitis,renal disease, acute kidney injury, liver injury, acute circulatoryfailure, surgery-induced inflammation, abscess-induced inflammation,multiple sclerosis, preeclampsia, cancer, cancer metastasis, tumorinvasion, peripheral artery disease, type 1 or type 2 diabetes,atherosclerotic cardiovascular disease, intermittent claudication,critical limb ischemic disease, myocardial infarction, carotidocclusion, umbilical cord occlusion, low birth-weight, premature birth,pulmonary insufficiency, peripheral neuropathy, hypoxic ischemia,bacterial infection, wounds, burns, lacerations, contusions, bonefractures, surgical procedures, tissue ischemia, rheumatoid arthritis,meningitis, inflammatory bowel disease, chronic obstructive pulmonarydisease, rhinitis, preterm labor, or an infectious disease.

In some embodiments, the infection is caused by a gram negativebacteria, such as Neisseria species including Neisseria gonorrhoeae andNeisseria meningitidis, Branhamella species including Branhamellacatarrhalis, Escherichia species including Escherichia coli,Enterobacter species, Proteus species including Proteus mirabilis,Pseudomonas species including Pseudomonas aeruginosa, Pseudomonasmallei, and Pseudomonas pseudomallei, Klebsiella species includingKlebsiella pneumoniae, Salmonella species, Shigella species, Serratiaspecies, Acinetobacter species; Haemophilus species includingHaemophilus influenzae and Haemophilus ducreyi, Brucella species,Yersinia species including Yersinia pestis and Yersinia enterocolitica,Francisella species including Francisella tularensis, Pasteurellaspecies including Pasteurella multocida, Vibrio cholerae, Flavobacteriumspecies, meningosepticum, Campylobacter species including Campylobacterjejuni, Bacteroides species (oral, pharyngeal) including Bacteroidesfragilis, Fusobacterium species including Fusobacterium nucleatum,Calymmatobacterium granulomatis, Streptobacillus species includingStreptobacillus moniliformis, and Legionella species includingLegionella pneumophila.

In a third aspect, featured is a kit for quantifying IAIP or anIAIP-IAIP ligand complex in a sample, in which the kit comprises an IAIPbinding agent and an IAIP detection agent and, optionally, one or moreof the following: a wash buffer, a blocking agent, a substrate fordetection of a label, and instructions for quantifying a level of IAIPor an IAIP-IAIP ligand complex in a sample. One or more of thecomponents of the kit may be provided in a container, such as a tube orvial, and/or in a form ready for use (e.g., application to a support ofthe kit (e.g., a plate or test strip).

In some embodiments, the binding agent is immobilized on a support.

In some embodiments, the detection agent is labeled.

In some embodiments, the IAIP binding agent is an IAIP-specific antibodyor an IAIP ligand.

In some embodiments, the kit further contains an IAIP ligand bindingagent. In some embodiments, the IAIP ligand binding agent is an antibodythat binds to an IAIP ligand.

In some embodiments, the IAIP detection agent is an IAIP-specificantibody or an IAIP ligand.

In some embodiments, the kit further contains an IAIP ligand detectionagent. In some embodiments, the IAIP ligand detection agent is anantibody that binds specifically to an IAIP ligand.

In some embodiments, the IAIP-specific antibody is a monoclonalantibody. In some embodiments, the monoclonal antibody is MAb 69.26 orMAb 69.31.

In some embodiments, the support is a plate, a resin, a container, amembrane, a biochip, a particle, a nanoparticle, a test strip, or abead.

In some embodiments, the label is an enzyme, an enzyme substrate,biotin, a particle, a fluorescent dye, a luminescent compound, or aradiolabel.

In some embodiments, the IAIP ligand is selected from the groupconsisting of endotoxin (LPS), heparin, a histone, hyaluronic acid,laminin, tenascin C, aggrecan, vitronectin, fibronectin, von WillebrandFactor, pentraxin-3 (PTX3), TNF-stimulated gene-6 (TSG-6), factor IX, acomplement component, factor XIIIa, and tissue transglutaminase.

Definitions

As used herein, the term “about” refers to a value that is no more than10% above or below the value being described. For example, the term“about 5 nM” indicates a range of from 4.5 nM to 5.5 nM.

As used herein, “administration” refers to providing or giving a subjecta therapeutic agent (e.g., IAIP), by any effective route. Exemplaryroutes of administration are described herein below.

As used herein, the term “antibody” (Ab) refers to an immunoglobulinmolecule that specifically binds to, or is immunologically reactivewith, a particular antigen, and includes at least the variable domain ofa heavy chain, and normally includes at least the variable domains of aheavy chain and of a light chain of an immunoglobulin. Antibodies andantigen-binding fragments, variants, or derivatives thereof include, butare not limited to, polyclonal, monoclonal, multispecific, human,humanized, primatized, or chimeric antibodies, heteroconjugateantibodies (e.g., bi-tri- and quad-specific antibodies, diabodies,triabodies, and tetrabodies), single-domain antibodies (sdAb),epitope-binding fragments, e.g., Fab, Fab′ and F(ab′)₂, Fd, Fvs,single-chain Fvs (scFv), rIgG, single-chain antibodies, disulfide-linkedFvs (sdFv), fragments comprising either a V_(L) or V_(H) domain,fragments produced by an Fab expression library, and anti-idiotypic(anti-Id) antibodies. Antibody molecules of the invention can be of anytype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2,IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.Moreover, unless otherwise indicated, the term “monoclonal antibody”(mAb) is meant to include both intact molecules as well as antibodyfragments (such as, for example, Fab and F(ab′)₂ fragments) that arecapable of specifically binding to a target protein. Fab and F(ab′)₂fragments lack the Fc fragment of an intact antibody, clear more rapidlyfrom the circulation of the animal, and may have less non-specifictissue binding than an intact antibody.

The term “antigen-binding fragment,” as used herein, refers to one ormore fragments of an antibody that retain the ability to specificallybind to a target antigen. The antigen-binding function of an antibodycan be performed by fragments of a full-length antibody. The antibodyfragments can be a Fab, F(ab′)₂, scFv, SMIP, diabody, a triabody, anaffibody, a nanobody, an aptamer, or a domain antibody. Examples ofbinding fragments encompassed of the term “antigen-binding fragment” ofan antibody include, but are not limited to: (i) a Fab fragment, amonovalent fragment consisting of the V_(L), V_(H), C_(L), and C_(H)1domains; (ii) a F(ab′)₂ fragment, a bivalent fragment comprising two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the V_(H) and C_(H)1 domains; (iv) a Fv fragmentconsisting of the V_(L) and V_(H) domains of a single arm of anantibody, (v) a dAb including V_(H) and V_(L) domains; (vi) a dAbfragment (Ward et al., Nature 341:544-546, 1989), which consists of aV_(H) domain; (vii) a dAb which consists of a V_(H) or a V_(L) domain;(viii) an isolated complementarity determining region (CDR); and (ix) acombination of two or more isolated CDRs which may optionally be joinedby a synthetic linker. Furthermore, although the two domains of the Fvfragment, V_(L) and V_(H), are coded for by separate genes, they can bejoined, using recombinant methods, by a linker that enables them to bemade as a single protein chain in which the V_(L) and V_(H) regions pairto form monovalent molecules (known as single-chain Fv (scFv); see,e.g., Bird et al., Science 242:423-426, 1988, and Huston et al., Proc.Natl. Acad. Sci. USA 85:5879-5883, 1988). These antibody fragments canbe obtained using conventional techniques known to those of skill in theart, and the fragments can be screened for utility in the same manner asintact antibodies. Antigen-binding fragments can be produced byrecombinant DNA techniques, enzymatic or chemical cleavage of intactimmunoglobulins, or, in some embodiments, by chemical peptide synthesisprocedures known in the art.

As used herein, the term “inter-alpha inhibitor proteins” or “IAIPs”refers to large, multi-component glycoproteins in a family ofstructurally related immunomodulatory proteins. IAIPs have been shown tobe important in the inhibition of an array of proteases includingneutrophil elastase, plasmin, trypsin, chymotrypsin, Granzyme K,preprotein convertase, furin, cathepsin G, and acrosin. IAIP exert abroad range of anti-inflammatory mechanisms, in addition to serineprotease inhibitory activity, such as binding to and inactivatingcomplement, extracellular histones, and coagulation factors, downregulating pro-inflammatory cytokines such as TNF-a and IL-6, downregulating adhesion factors such as VCAM and ICAM, and down regulatingNFkB. IAIP are also importantly involved in promoting protection andrepair of tissues where the heavy chains are transferred to matrixproteins to promote cellular migration and proliferation. In humanplasma, IAIPs are found at relatively high concentrations (400-800mg/L). Unlike other inhibitor molecules, this family of inhibitorstypically includes a combination of polypeptide chains (light and heavychains) covalently linked by a chondroitin sulfate chain. The heavychains of IAIPs (H1, H2, and H3) are also called hyaluronic acid (HA)binding proteins. The major forms of IAIPs found in human plasma areinter-alpha-inhibitor (IαI), which contains two heavy chains (H1 and H2)and a single light chain (L), and pre-alpha-inhibitor (PαI), whichcontains one heavy (H3) and one light chain (L). Another IAIP is thelight chain (also termed bikunin (bi-kunitz inhibitor) with two Kunitzdomains), bound to the glycosaminoglycan, which is known to broadlyinhibit plasma and tissue serine proteases. Another IAIP is the heavychain-related molecule H4, which circulates in the blood without linkageto bikunin. Yet another IAIP is the heavy chain-related molecule H5. IαIand PαI present in the plasma fraction have an apparent molecular weightof between about 60 kDa to about 280 kDa.

As used herein, the term “IAIP ligand” refers to a molecule or afragment thereof that binds to IAIP in vivo or in vitro (e.g., endotoxin(LPS), heparin, a histone, hyaluronic acid, matricellular proteins(e.g., vitronectin, fibronectin, tenascin C, laminin, aggrecan), vonWillebrand Factor, pentraxin-3 (PTX3), TNF-stimulated gene-6 (TSG-6),coagulation factors (e.g., factor IX and factor XIIIa), a complementcomponent, divalent cations such as Ca²⁺, and tissue transglutaminase).IAIP ligands also include molecules that are predicted to bind to IAIPbased on domain structure (e.g., proteins with RGD domains that wouldbind to the von Willebrand factor A domain of the IAIP heavy chains).The IAIP ligands for use in the methods described herein include ligandsthat bind to the heavy chain of IAIP, to the IAIP complex, or to the GAGof IAIP (e.g., ligands that do not bind solely to bikunin).

As used herein, the term “IAIP-specific antibody” or “antibody thatspecifically binds to IAIP” refers to any protein or peptide-containingmolecule that includes at least a portion of an immunoglobulin molecule,such as at least one complementarity determining region (CDR) of a heavyor light chain or a ligand binding portion thereof, a heavy chain orlight chain variable region, a heavy chain or light chain constantregion, a framework region, or any portion thereof, that is capable ofbinding IAIP and that does not specifically bind to any other protein.An antibody that binds specifically to IAIP will bind to IAIP andprovide a signal that is it least twice the background signal or noise,and more typically more than 10 to 100 times background.

As used herein, the term “monoclonal antibody” refers to an antibodythat is derived from a single clone, including any eukaryotic,prokaryotic, or phage clone, and not the method by which it is produced.

As used herein, a “pharmaceutical composition” or “pharmaceuticalpreparation” is a composition or preparation having pharmacologicalactivity or other direct effect in the mitigation, treatment, orprevention of disease, and/or a finished dosage form or formulationthereof. The composition is, for example, indicated for human use (e.g.,according to drug or biologic regulatory guidelines, such as thosepromulgated by the F.D.A. and/or the E.M.A.).

As used herein, the term “pharmaceutically acceptable” refers to thosecompounds, materials, compositions and/or dosage forms, that aresuitable for contact with the tissues of a subject, such as a mammal(e.g., a human) without excessive toxicity, irritation, allergicresponse, and other problem complications commensurate with a reasonablebenefit/risk ratio.

As used herein, the phrase “reducing the likelihood of developing”refers to prophylactic treatment of a patient (e.g., a human) who issusceptible to, or otherwise at risk of, a particular disease, syndrome,or condition (e.g., the conditions described herein, such as aninflammatory disease or an infection)) or is at risk of an increase inthe degree or severity of a current disease, syndrome, or condition, forexample, a patient having community acquired pneumonia (CAP) who is atrisk of progressing to severe community acquired pneumonia (sCAP).

As used herein, the term “reference” refers to a parameter (e.g.,protein level, concentration, nucleic acid expression level, and genecopy number) of a sample or standard that is used for comparisonpurposes to the parameter in a test sample. For example, a referencesample can be obtained from a healthy individual (e.g., an individualwho does not have an inflammatory disease or infection). A referencelevel can be the level (or an average thereof) of expression orconcentration of an analyte (e.g., protein (e.g., an IAIP), nucleicacid, carbohydrate, etc.) determined from one or more reference samples.For example, the reference can be an average level of an analyte (e.g.,IAIP) (e.g., a mean level or median level) among a plurality ofindividuals (e.g., healthy individuals, or individuals who do not havean inflammatory disease or infection). In other instances, a referencelevel can be a predetermined threshold level, e.g., based on level orconcentration of an analyte as otherwise determined, e.g., by empiricalassays.

As used herein, the term “sample” refers to a specimen (e.g., blood,blood component (e.g., serum or plasma), urine, saliva, amniotic fluid,lung lavage, cerebrospinal fluid, tissue (e.g., tissue biopsy or tissuehomogenate), pancreatic fluid, synovial fluid, and cells) isolated froma subject (e.g., a mammal, such as a human).

As used herein, the phrase “specifically binds” refers to a bindingreaction which is determinative of the presence of an analyte (e.g., aprotein, such as IAIP) in a heterogeneous population of proteins andother biological molecules. The analyte may be an antigen that isrecognized, e.g., by an antibody or antigen-binding fragment thereof.Specific binding between an analyte to be measured and a binding agent(e.g., an antibody or antigen-binding fragment or ligand) exhibits aK_(D) of less than 100 nM. For example, an antibody or antigen-bindingfragment thereof that specifically binds to an antigen (e.g., IAIP)binds to the antigen with a K_(D) of up to 100 nM (e.g., between 1 pMand 100 nM). An antibody or antigen-binding fragment thereof that doesnot exhibit specific binding to a particular antigen or epitope thereof(e.g., IAIP) exhibits a K_(D) of greater than 100 nM (e.g., greater than500 nm, 1 μM, 100 μM, 500 μM, or 1 mM) for that particular antigen orepitope thereof. A variety of immunoassay formats may be used to selectantibodies specifically immunoreactive with a particular protein orcarbohydrate. For example, solid-phase ELISA immunoassays are routinelyused to select antibodies specifically immunoreactive with a protein orcarbohydrate. See, Harlow & Lane, Antibodies, A Laboratory Manual, ColdSpring Harbor Press, New York (1988) and Harlow & Lane, UsingAntibodies, A Laboratory Manual, Cold Spring Harbor Press, New York(1999), for a description of immunoassay formats and conditions that canbe used to determine specific immunoreactivity.

As used herein, the term “sepsis” refers to a systemic response to aninfection (referred to herein as “infectious sepsis”) or to anon-infectious process associated with acute tissue injury and innateimmune activation (referred to interchangeably herein as “sterileinflammation” or “sterile sepsis”), which can lead to tissue damage,organ failure, and death. Infectious sepsis can result from an infectioncaused by bacteria, viruses, fungi, or other microorganisms such asparasites (e.g., protozoan parasites). Sterile sepsis can occur afterhemorrhagic shock, polytrauma, pancreatitis, transplant rejection,autoimmune disease, inorganic compounds, crystals, chemicals, orischemia/reperfusion and is not associated with the presence of a knowninfection.

As used herein, the term “subject” refers to a mammal, including, butnot limited to, a human or non-human mammal, such as a primate, bovine,equine, porcine, ovine, feline, or canine. The subject may be a patient.

As used herein, the term “treating” refers to reducing or ameliorating adisorder and/or symptoms associated therewith. It will be appreciatedthat, although not precluded, treating a disorder or condition does notrequire that the disorder or symptoms associated therewith be completelyeliminated.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic depicting the structure of circulating (e.g., inblood) IAIP (IaI and PaI) and free light chain (LC, bikunin), forexample, excreted in urine. Heavy and light chains of IAIP are uniquelylinked by glycosaminoglycan (GAG).

FIG. 2A is a schematic depicting a competitive IAIP ELISA assay. In thisassay, purified IAIP is immobilized to a support, such as a multi-wellplate, and a biological sample and a labeled antibody directed to IAIP(e.g., MAb 69.26) are then added to the purified IAIP.

FIG. 2B is a graph showing a standard curve of data produced by acompetitive IAIP ELISA assay. This assay provides an indirect measure ofIAIP based on competitive antibody binding between IAIP in the sampleand immobilized, purified IAIP. Lower signal indicates higher amounts ofIAIP in the sample.

FIG. 3A is a schematic depicting the “sandwich-type” ELISA using labeledIAIP ligands (e.g., biotinylated heparin or LPS) and exemplary standardcurves generated using said ELISA. In this version of the assay, anantibody specific for IAIP (e.g., MAb 69.26) is immobilized to asupport, such as a multi-well plate, and a biological sample is thenadded to the support containing the immobilized antibody. If IAIP ispresent in the sample, it will bind to the antibody and then be detectedby the addition of the labeled IAIP ligand.

FIGS. 3B and 3C are graphs showing standard curves for heparin-IAIP andendotoxin (LPS)-IAIP sandwich-type ELISAs, respectively. This assayprovides a direct measure of IAIP concentration, with increasing signalindicating higher amounts of IAIP in the sample.

FIG. 4 is a graph showing measurements of IAIP concentration obtainedusing a competitive ELISA as depicted in FIG. 2A. IAIP was measured inplasma samples from patients with severe community acquired pneumonia(sCAP) on successive days during hospitalization and compared to IAIPlevels in normal control subjects. The competitive ELISA yielded anaverage IAIP concentration of 250 μg/mL in patients with sCAP and 330μg/mL in healthy controls, and found significant differences betweenIAIP levels in subjects with sCAP and healthy controls on days 0, 1, and3.

FIG. 5 is a graph showing measurements of IAIP concentration obtainedusing a “sandwich-type” ligand-IAIP ELISA as depicted in FIG. 3A. Theresults depicted in FIG. 5 were generated using heparin as the IAIPligand. The same samples evaluated using the competitive ELISA in FIG. 4were also measured using this assay. The heparin-IAIP ELISA yielded anaverage IAIP concentration between 125 and 150 μg/mL in patients withsCAP and 422 μg/mL in healthy controls, and found differences betweenIAIP levels in subjects with sCAP and healthy controls that were morestatistically significant than the differences observed using thecompetitive ELISA at all time points.

FIG. 6 is a graph showing measurements of IAIP concentration obtainedusing a “sandwich-type” ligand-IAIP ELISA as depicted in FIG. 3A. Theresults depicted in FIG. 6 were generated using LPS as the IAIP ligand.The same samples evaluated using the competitive ELISA in FIG. 4 and theheparin-IAIP ELISA in FIG. 5 were also measured using this assay. TheLPS-IAIP ELISA yielded an average IAIP concentration between 118 and 145μg/mL in patients with sCAP and 338 μg/mL in healthy controls, and founddifferences between IAIP levels in subjects with sCAP and healthycontrols that were more statistically significant than the differencesobserved using the competitive ELISA at all time points. The LPS-IAIPELISA performed comparably to the heparin-IAIP ELISA in terms ofincreased sensitivity and a more measurable difference between IAIPlevels in subjects with sCAP and healthy controls.

FIG. 7 is a series of blots showing the binding of heparin and anIAIP-specific antibody (MAb 69.26) to IAIP. As shown in the middle blot,biotinylated heparin bound to purified IAIP (250 kDa IαI and 125 kDaPαI), but did not bind to the IAIP light chain, bikunin, or to thenegative control human serum albumin. In contrast, MAb 69.26 bound toboth purified IAIP and bikunin. These data suggest that heparin binds tothe heavy chain of IAIP, which may lead to a more accurate measure ofcirculating, intact IAIP in the heparin-IAIP ELISA.

FIGS. 8A-8C are a series of graphs showing measurements of IAIPconcentration obtained using a competitive ELISA as depicted in FIG. 2Aand “sandwich-type” ligand-IAIP ELISAs as depicted in FIG. 3A. IAIP wasmeasured in plasma samples from patients with severe pneumonia, severesepsis, and in normal control subjects. Shown below each graph is themean±SEM of IAIP concentration and the number of patient samples tested(in brackets). As shown in FIG. 8A, the competitive ELISA yielded anaverage IAIP concentration of 246 μg/mL in patients with severepneumonia, 250 μg/mL in patients with severe sepsis, and 330 μg/mL inhealthy controls, and found significant differences between IAIP levelsin subjects with severe pneumonia and healthy controls, but not betweensubjects with severe sepsis and healthy controls. As shown in FIG. 8B,the LPS-IAIP ELISA yielded an average IAIP concentration of 141 μg/mL inpatients with severe pneumonia, 150 μg/mL in patients with severesepsis, and 338 μg/mL in healthy controls, and found significantdifferences between IAIP levels in subjects with severe pneumonia orsevere sepsis and healthy controls. As shown in FIG. 8C, theheparin-IAIP ELISA yielded an average IAIP concentration of 145 μg/mL inpatients with severe pneumonia, 193 μg/mL in patients with severesepsis, and 422 μg/mL in healthy controls, and found significantdifferences between IAIP levels in subjects with severe pneumonia orsevere sepsis and healthy controls. The LPS-IAIP ELISA performedcomparably to the heparin-IAIP ELISA in terms of having increasedsensitivity and providing more statistically significant resultscompared to the competitive ELISA.

FIG. 9 is a histogram showing the binding of biotinylated LPS toimmobilized plasma-derived IAIP, bovine serum albumin (BSA), and IgG ofIAIP-specific antibody MAb 69.26. LPS demonstrated substantial bindingto IAIP and little to no binding to MAb 69.26, which served as anegative control. Notably, LPS did not exhibit binding to the IAIP lightchain, bikunin, indicating that the heavy chain of IAIP may facilitatebinding to LPS.

FIG. 10 is a histogram showing the binding of biotinylated IAIP to LPS,BSA, IgG of IAIP-specific antibody MAb 69.26, and non-fat dried milk. Asexpected, IAIP bound to MAb 69.26 and LPS and showed minimal binding toBSA and non-fat dried milk. These data confirm the results of FIG. 9.

FIG. 11 is a histogram showing the effect of pH on the binding ofbiotinylated IAIP to LPS, BSA, and IgG of IAIP-specific antibody MAb69.26. These data demonstrate that IAIP bound most strongly to LPS at pH5, with reduced binding at pH 6 and pH 7, and little or no binding at pH8 or higher or pH 4 or lower. In contrast, IAIP bound to IgG of MAb69.26 at pH 5-pH 9, but did not bind at pH 4 or below. As in previousexperiments, IAIP did not bind to BSA.

FIG. 12 is a histogram showing the effect of salt (NaCl) concentrationon the binding of biotinylated IAIP to LPS, BSA, and IgG ofIAIP-specific antibody MAb 69.26. These data show that the binding ofIAIP to both LPS and MAb 69.26 was unaffected by salt, indicating strongand specific binding. No binding to BSA was observed at any of the saltconcentrations tested.

FIG. 13 is a histogram showing the effect of non-ionic detergent NP-40on the binding of biotinylated IAIP to LPS, BSA, and IgG ofIAIP-specific antibody MAb 69.26. Binding of IAIP to LPS was enhanced bythe addition of 0.05% NP-40, and was still observed in the presence of1% NP-40, indicating a strong binding interaction. No binding to BSA wasobserved at any of the NP-40 concentrations tested.

FIG. 14 is a histogram showing the effect of non-ionic detergentTween-20 on the binding of biotinylated IAIP to LPS, BSA, and IgG ofIAIP-specific antibody MAb 69.26. Binding of IAIP to LPS was enhanced bythe addition of 0.05% Tween-20, and was still observed in the presenceof 1% Tween-20, indicating a strong binding interaction. No binding toBSA was observed at any of the Tween-20 concentrations tested.

FIGS. 15A-15B are histograms depicting blood IAIP level (FIG. 15 A) andCRP (FIG. 15 B) in infants with NEC (n=14) and SIP (n=13) as well ingender, weight and gestational age-matched healthy controls (n=26) atinitial presentation. Decreased IAIP levels were found in infants withproven NEC (mean±SD: 139±21 ug/mL) while the levels in healthy controls(276±110 ug/mL) or infants with SIP (319±72 ug/mL) were significantlyhigher (p<0.05 and p<0.005). In contrast, no statistically significantdifference between IAIP levels in infants with SIP and controls (p>0.4).When CRP levels were tested, no significant difference was found betweenSIP, NEC and control group (p>0.05).

FIGS. 16A-16B are graphs depicting ROC curves of IAIP and CRP in NECinfants. The predictive value of IAIP level is superior with sensitivityof 100%, specificity of 88.2%, PPV 41% and NPV 100%, (FIG. 16A) comparedto CRP (FIG. 16B) with sensitivity of 100%, specificity of 64.7%, PPV18% and NPV 98%.

FIG. 17 is a graph depicting the result of longitudinal studies ofplasma IAIP levels in infants with NEC (n=8) and SIP (n=9). Blood wascollected serially at the time before and after the onset of the diseasepresentation. No significant difference in IAIP levels was found in bothinfant groups with SIP and NEC before the disease onset (p value>0.6),but IAIP levels in infants with NEC were significantly lower comparedwith the levels in infants with SIP (p<0.04) up to 10 days after theonset.

FIG. 18 is a schematic of a lateral flow immunoassay-based IAIP rapidtest.

FIG. 19 is a graph depicting the standard curve of the rapid IAIP testusing an ESEQuant reader. The value of each points were plotted as themean+SD of a total of 13 independent analysis. The rapid test standardcurve is suitable for plasma samples with IAIP level that ranges from17.5 to 1100 μg/mL.

FIGS. 20A-20C are a series of graphs depicting the correlations betweenthe results obtained by an IAIP rapid test using ESEQuant reader, Detektreader, iCalQ reader and the results obtained by the establishedcompetitive ELISA format.

FIGS. 21A-21B are a series of graphs depicting the quantification ofIAIP using a “sandwich-type” ELISA in which hyaluronic acid isimmobilized on a 96-well plate to capture IAIP, and IAIP is detectedusing biotin-conjugated MAb 69.26 (a monoclonal antibody against humanIAIP). The assay can be used to quantify IAIP in serial dilutions ofboth human plasma (FIG. 21A) and purified IAIP (FIG. 21B) using 50 ng,100 ng, or 200 ng hyaluronic acid per well.

DETAILED DESCRIPTION

Featured are methods of measuring IAIP concentration in a sample (e.g.,a sample from a subject, such as a blood sample) using a reagent thatcan be used to measure the amount of IAIP in the sample (e.g., a reagentthat directly binds to IAIP in the sample, such an IAIP ligand, or areagent that binds to an IAIP ligand that is bound to IAIP). The reagentcan be measured by using a detectable label. The methods can be used toidentify subjects having or at risk of developing an inflammatorydisease or condition or an infection, to determine a health status of ordisease severity in a subject, and to monitor treatment of a conditionin a subject (e.g., an inflammatory disease or condition or aninfection) administered a therapeutic agent (e.g., IAIP or an agent fortreating an inflammatory disease or condition or an infection).

Assays for Quantification of IAIP

Provided are methods of quantifying IAIP in a sample(s) collected from asubject. Previous methods of detecting IAIP relied on antibody-basedcompetitive ELISAs, which provide an indirect measure of IAIPconcentration in a sample. This method detects both intact IAIPcomplexes (e.g., IAIP containing one or more heavy chains (H1-H5) andthe light chain, bikunin) and bikunin alone. In contrast, the methodsdescribed herein use a reagent (e.g., an IAIP ligand) that detects IAIPin the sample directly by binding to an IAIP heavy chain, a heavy andlight chain of an intact IAIP complex, or a GAG of an IAIP complex, butdo not bind bikunin alone.

Without wishing to be bound by theory, the IAIP ligand-based assaysdescribed herein exhibit greater sensitivity and robustness than acompetitive ELISA due to the detection of the IAIP heavy chain or intactIAIP complexes. The present methods provide an improved readout of theamount of functional IAIP in a sample relative to antibody-based assaysthat detect cleaved or degraded IAIP light chain.

The methods described herein include the use of an IAIP ligand to detectIAIP, e.g., IAIP captured on a substrate (e.g., a solid support, e.g., aplate, a resin, a particle, a container, a membrane, or a bead), using,e.g., an IAIP-specific agent, such as an antibody, the use of an IAIPligand to capture IAIP for subsequent detection with an IAIP-specificantibody, and the use of an IAIP ligand to capture IAIP for subsequentdetection with a second, different IAIP ligand.

A first method that can be used to detect intact IAIP in a sampleinvolves an IAIP binding agent (e.g., an IAIP-specific antibody or anIAIP ligand) that is attached to a support and used to capture IAIP inthe sample. An IAIP detection agent (e.g., an IAIP ligand or anIAIP-specific antibody) is then added that binds to the IAIP that hasformed a complex with the IAIP binding agent. IAIP concentration canthen be quantified, e.g., by detecting the presence of a label, which iseither attached directly to the IAIP detection agent or is attached to areagent that binds to the IAIP detection agent.

A second method that can be used to detect an amount of IAIP in a sampleinvolves detecting IAIP-IAIP ligand complexes that have formed in vivoand that are present in the sample. This method also begins with the useof a binding agent that is attached to a support. The binding agent canbind to either IAIP or the IAIP ligand. A detection agent is then added,which binds to the other component of the IAIP-IAIP ligand complex(e.g., if the binding agent is directed to IAIP, the detection agent isdirected to the IAIP ligand, and, conversely, if the binding agent isdirected to the IAIP ligand, the detection agent is directed to IAIP).The concentration of the IAIP can then be measured, e.g., by detectingthe presence of a label, which is either attached directly or indirectlyto the detection agent.

The reagents used in the methods and the method steps are discussed indetail below.

IAIP Ligands

IAIP ligands for use in the methods described herein include ligandsthat bind to one or more IAIP heavy chains, to an IAIP heavy chain(s)and the light chain, bikunin (e.g., intact IAIP), or to a GAG of IAIP(e.g., a GAG in intact IAIP). Intact IAIP is a complex that contains atleast one IAIP heavy chain (H1, H2, H3, H4, and/or H5) and the IAIPlight chain (bikunin). Any ligand that binds to a heavy chain of IAIP orintact IAIP can be used in the methods described herein, including,e.g., endotoxin (lipopolysaccharide, LPS), heparin, histone, hyaluronicacid, vitronectin, fibronectin, tenascin C, laminin, aggrecan, vonWillebrand Factor, pentraxin-3 (PTX3), TNF-stimulated gene-6 (TSG-6),coagulation proteins (e.g., factor IX and factor XIIIa), complementproteins (e.g., C1q, C2, C3, C4, C5, C6, C8, properdin, and factor D),divalent cations (e.g., Ca²⁺), and tissue transglutaminase. IAIP ligandscan be labeled according to standard techniques known in the art (e.g.,using one or more of the detectable labels described below).

IAIP Antibodies

Antibodies that specifically bind to IAIP can be used as binding agentsor detection agents in the methods described herein. Antibodies thatspecifically bind to IAIP are antibodies or antigen binding fragmentsthereof that do not specifically bind to any proteins other than IAIP(e.g., interaction of an IAIP-specific antibody with non-IAIP proteinsyields a signal similar to background). The antibody that bindsspecifically to IAIP can bind to a heavy chain of IAIP, the light chainof IAIP, or both, or to a GAG of IAIP. Preferably, the antibody binds toa heavy chain of IAIP or to an intact IAIP containing heavy and lightchains. The antibody may be raised against human IAIP, or against IAIPfrom another mammal (e.g., non-human primate, cow, pig, sheep, goat,cat, dog, rat, mouse, rabbit, guinea pig, or any other non-human mammalthat expresses IAIP). The antibody that binds specifically to IAIP maybind only human IAIP, or may be capable of binding IAIP from humans andother mammals. The antibody can be produced by immunization of an animaltypically used to generate antibodies with IAIP (e.g., rabbit, guineapig, rat, mouse, sheep, donkey, goat, hamster, and chicken). TheIAIP-specific antibody can be polyclonal (e.g., PAb R16, PAb R20, PAbR21), monoclonal (e.g., MAb 69.26 or MAb 69.31), chimeric, orrecombinant.

Labels

Labels for detecting the concentration of IAIP in the methods describedherein can be attached or conjugated to IAIP ligands, IAIP-specificantibodies, or to other reagents described herein. Labels suitable fordetection of IAIP ligands, IAIP ligand-specific antibodies, and/orIAIP-specific antibodies include biotin, enzymes (e.g., horseradishperoxidase (HRP), alkaline phosphatase (AP), β-galactosidase,acetylcholinesterase, and catalase), enzyme substrates, radiolabels(e.g., radioisotopes), luminescent compounds, particles (e.g., colloidalgold (e.g., gold nanoparticles), a magnetic particle, or a latexparticle) and fluorescent dyes. The label can then be assessed directly(e.g., through imaging of fluorescent dyes, detection of radioactivity,or detection of particles), through the use of enzyme-conjugated avidinor streptavidin (e.g., avidin or streptavidin conjugated to AP or HRPfor detection of biotin), and/or through use of a substrate, and can bevisualized by known methods and devices, including, e.g., aspectrophotometer, fluorometer, luminometer, or liquid scintillationcounter. Substrates may be colorimetric (e.g., PNPP for detecting AP; orABTS, OPD, or TMB for detecting HRP), chemiluminescent, or fluorescent.Substrates also include liquid scintillators for detection ofradioactivity. Standard detection methods known in the art can be usedto detect the labels described herein.

Samples

The methods described herein can be performed using a sample from asubject (e.g., a human subject). Suitable samples include fluid samples.For example, IAIP can be measured in a sample of blood or plasma from asubject (e.g., a subject with an inflammatory disease or condition, suchas sepsis, or a subject at risk for developing an inflammatory diseaseor condition, such as sepsis). The methods described herein can also beperformed using a sample of other bodily fluids, such as urine,cerebrospinal fluid, synovial fluid, amniotic fluid, interstitial fluid,follicular fluid, peritoneal fluid, bronchoalveolar lavage fluid, breastmilk, sputum, lymph, and bile. Tissue samples (e.g., biopsies) from asubject can be homogenized in an appropriate buffer to create a “fluid”for quantification of IAIP using these methods.

The volume of sample used in the assay will vary depending on the typeof assay being performed (e.g., an ELISA, a lateral flow immunoassay, oranother assay) and the support (e.g., a plate, a membrane, a test strip,or another support). The volume of sample used can be from about 1 μL toabout 500 μL (e.g., from about 1 μL to about 150 μL, e.g., from 1 μL toabout 30 μL for an assay such as a lateral flow immunoassay, or fromabout 50 μL to about 200 μL for an assay such as an ELISA). The samplecan be diluted before use in the assay with a buffer that will notinterfere with binding to the binding or detection agent (e.g., water,PBS, or a buffer used in the methods of the assay), and may be diluted1:2, 1:3, 1:4, 1:5, 1:10, 1:15, 1:20, 1:100 or more.

Method for Detecting Intact IAIP

A first IAIP quantification assay involves contacting a samplecontaining IAIP to an IAIP binding agent (e.g., an antibody thatspecifically binds to IAIP or an IAIP ligand) to form an IAIP-bindingagent complex. The IAIP binding agent may be attached to a support(e.g., a solid support). Suitable supports include plates (e.g.,multi-well plates), particles (e.g., magnetic particles, nanoparticles,magnetic nanoparticles), biochips, resins, membranes (e.g.,nitrocellulose membranes, PVDF membranes), containers (e.g., tubes),test strips (e.g., cellulose, glass fiber, nitrocellulose), and beads(e.g., protein A or protein G beads, magnetic beads, glass beads,plastic beads). The support is preferably capable of being washed one ormore times (e.g., using a buffer, such as TBS, TBS-T, PBS, or PBS-T) toremove material that does not bind to the IAIP binding agent.

The IAIP-binding agent complex is then contacted with an IAIP detectionagent (e.g., an IAIP ligand or an antibody that specifically binds toIAIP). The IAIP detection agent may be conjugated to a label (e.g., oneor more of the labels described above), which can then be detected usingknown detection methods. Alternatively, the IAIP detection agent can bedirectly detected without the use of a label. After the addition of theIAIP detection agent, an additional wash step (e.g., one or more) can beperformed to remove unbound detection agent.

IAIP can then be measured based on signal from the conjugated label orthe attached detection agent (e.g., enzyme activity or fluorescence)using standard techniques known in the art. If an enzyme is used as thelabel, substrate can be added to produce the signal (for example, acolor change) and can be read by a device suitable for detecting thesignal, such as a spectrophotometer. The signal (for example, absorbanceor fluorescence) can be plotted against a standard with knownconcentration of IAIP to establish a standard curve or can be comparedagainst a known reference concentration. The unknown concentration inthe samples can be calculated and determined based on the establishedstandard curve or reference concentration value.

Before the addition of the sample to the IAIP binding agent, a blockingstep can be performed to prevent or reduce non-specific binding.Blocking agents for use in the methods described herein include, e.g.,milk, BSA, casein, gelatin (e.g., fish gelatin), and serum (e.g., goatserum, donkey serum, horse serum, fetal bovine serum), among others.

The method steps can be performed at a pH of 7.0 to 3.5 (e.g., pH 5.0 topH 3.5, e.g., pH 4.0). For example, either or both of the binding stepand the detection step can be performed at a pH of 7.0 to 3.5 (e.g., pH5.0 to pH 3.5, e.g., pH 4.0). The IAIP binding agent can be prepared ina buffer with a pH of 7.0 to 3.5 (e.g., pH 5.0 to pH 3.5, e.g., pH 4.0)and/or the IAIP detection agent (e.g., the IAIP ligand) can be preparedin a buffer with a pH of 7.0 to 3.5 (e.g., pH 5.0 to pH 3.5, e.g., pH4.0). A low pH buffer (e.g., pH of 5.0 or lower, e.g., pH 4.0) can beused to improve detection of IAIP.

Also, to stabilize IAIP during the assay, divalent cations (e.g., Ca2+,Mg2+, Mn2+, Fe2+, etc.) may be added to the sample before or duringcontact with the IAIP binding agent, and/or added to the buffercontaining the detection agent before or during contact with the bindingagent-IAIP complex. Divalent cations can be provided in a concentrationof 1 μM to 1 M (e.g., 100 μM to 100 mM, 1 mM to 10 mM).

a) IAIP-Specific Antibody Binding Agent and IAIP Ligand Detection Agent

In one example of the method described herein, the IAIP binding agent isan antibody that binds specifically to IAIP (e.g., MAb 69.26 or MAb69.31) and the detection agent is a labeled IAIP ligand (e.g., heparin,hyaluronic acid, or LPS). Alternatively, the ligand can be unlabeled anddetected using a labeled antibody that is specific for the IAIP ligand,or the unlabeled ligand can be detected using an unlabeled antibody thatis specific for the IAIP ligand, which is then detected using a labeledsecondary antibody (e.g., a labeled secondary antibody that binds to theantibody that is specific for the IAIP ligand but does not bind to theIAIP antibody, e.g., the IAIP antibody and the IAIP ligand antibody aregenerated in different host species). If the ligand is unlabeled anddetected through the use of a labeled ligand-specific antibody, or aligand-specific antibody and a labeled secondary antibody, additionalwash steps may be performed before and/or after incubation with eachantibody.

b) IAIP Ligand Binding Agent and IAIP-Specific Antibody Detection Agent

In another example of the method described herein, the IAIP bindingagent is an IAIP ligand (e.g., heparin, hyaluronic acid, or LPS), andthe detection agent is an antibody that is specific for IAIP (e.g., MAb69.26 or MAb 69.31). In this example, the antibody that is specific forIAIP can be conjugated directly to a label, or the antibody can bedetected using a labeled secondary antibody that binds to theIAIP-specific antibody. If a labeled secondary antibody is used, a washstep can be performed after incubation with the secondary antibody tominimize non-specific signal prior to IAIP measurement.

c) IAIP Ligand Binding Agent and IAIP Ligand Detection Agent

In a third example of the method described herein, the binding agent isan IAIP ligand (e.g., hyaluronic acid), and the detection agent is alabeled IAIP ligand that binds to a different region of IAIP (e.g.,heparin, hyaluronic acid, or LPS). This method can be performed with anytwo IAIP ligands that do not bind to the same region of IAIP (e.g., twoIAIP ligands that do not compete for binding to IAIP or for which thebinding of one of the IAIP ligands does not sterically hinder thebinding of the second IAIP ligand to IAIP). The ligand that acts as theIAIP detection agent can be unlabeled and detected using a labeledantibody that is specific for the IAIP ligand that acts as the detectionagent, or it can be detected by a labeled secondary antibody that bindsto an unlabeled antibody that is specific for the IAIP ligand that actsas the detection agent. If labeled antibodies are used for detection ofthe IAIP ligand that acts as the detection agent, additional wash stepsmay be performed before and/or after incubation with each antibody.

d) IAIP-Specific Antibody Binding Agent and IAIP-Specific AntibodyDetection Agent

In a fourth example of the method described herein, the binding agent isan IAIP-specific antibody and the detection agent is a labeledIAIP-specific antibody. In this example, at least one of theIAIP-specific antibodies is capable of binding to either intact IAIP orIAIP that includes at least one heavy chain (e.g., the antibody does notbind to bikunin unless at least one IAIP heavy chain is present, e.g.,the antibody does not bind to cleaved or degraded IAIP lacking bikunin).Also, the two IAIP-specific antibodies used in the assay bind differentepitopes of IAIP (e.g., the antibodies do not compete for binding toIAIP). If the two IAIP-specific antibodies are generated using differenthost species, then the IAIP-specific antibody used as the detectionagent may be unlabled and detected using a labeled secondary antibody.If a labeled secondary antibody is used, additional wash steps may beperformed before and/or after incubation with the labeled secondaryantibody.

e) Quantification of IAIP Using a Rapid Lateral-Flow Immunoassay (LFIA)

In a fifth example, the methods described herein can be performed usinga lateral-flow immunoassay-based test. In this example, a small volumeof a sample (e.g., 1-30 μL e.g., 15 μL) can be applied to a test strip(e.g., a cellulose, glass fiber, or nitrocellulose) in an undiluted formor in a diluted form (e.g., diluted 1:2, 1:5, 1:10, 1:20, 1:100 or more,e.g., with a buffer (e.g., PBS) or water) and a buffer is then added topush the sample through the strip. The strip contains an IAIP bindingagent (e.g., an IAIP-specific antibody or an IAIP ligand) to bind toIAIP in the sample. IAIP can be detected using an IAIP detection agent(e.g., a labeled IAIP-specific antibody or a labeled IAIP ligand), whichcan be added to the strip before, after, or concurrent with the additionof the sample. The test strip can be read using an appropriate readerfor quantification of the label attached to the IAIP detection agent(e.g., a portable tabletop lateral flow reader, a handled PDA-basedreader, or a smartphone/tablet based reader, among others). Theselection of the binding and detection agent combination can be madebased on the parameters outlined herein. This assay can be performedquickly (e.g., in 15 minutes or less, such as 15, 10, or 7 minutes orless) and provides a quantitative and rapid measurement of IAIP in asmall sample volume.

Detection of Naturally Occurring IAIP-IAIP Ligand Complexes

Also provided are methods for detecting naturally occurring IAIP-IAIPligand complexes (e.g., IAIP-IAIP ligand complexes that have formed invivo in a subject (e.g., a human subject) and that are present in asample from the subject). This method involves contacting a sample(e.g., a fluid sample, such as plasma, serum, blood, bronchoalveolarfluid, cerebrospinal fluid, sputum, urine or other bodily fluids) to abinding agent, e.g., a binding agent that is attached to a support(e.g., a solid support). The binding agent can be an IAIP binding agent(e.g., an antibody that specifically binds to IAIP, for example, MAb69.26 or MAb 69.31, or an IAIP ligand), or the binding agent can be anantibody that specifically binds to the IAIP ligand of interest (e.g.,the IAIP ligand that is suspected to have formed an IAIP-IAIP ligandcomplex in vivo). Suitable supports include plates (e.g., multi-wellplates), particles (e.g., magnetic particles, nanoparticles, magneticnanoparticles), biochips, resins, containers (e.g., tubes), membranes(e.g., nitrocellulose membranes, PVDF membranes), test strips (e.g.,cellulose, glass fiber, or nitrocellulose) and beads (e.g., protein A orprotein G beads, magnetic beads, glass beads, plastic beads). Thesupport is preferably capable of being washed one or more times (e.g.,using a buffer, such as TBS, TBS-T, PBS, or PBS-T) to remove materialthat does not bind to the binding agent.

If the binding agent used in the assay is an IAIP binding agent (e.g.,an antibody that specifically binds to IAIP, for example, MAb 69.26 orMAb 69.31, or an IAIP ligand), the IAIP-IAIP ligand complex can bedetected using a detection agent that is an antibody directed againstthe IAIP ligand of interest by contacting the antibody to the IAIPcomplex-binding agent complex. If the binding agent used in the assay isan antibody that specifically binds to the IAIP ligand of interest, theIAIP-IAIP ligand complex can be detected using a detection agent thatspecifically binds to IAIP (e.g., an antibody that specifically binds toIAIP, for example, MAb 69.26 or MAb 69.31, or a different IAIP ligand)by contacting the detection agent to the IAIP complex-binding agentcomplex.

A wash step (e.g., one or more) can be performed after incubation withthe detection agent. Also, before the addition of the sample to the IAIPor IAIP ligand binding agent, a blocking step can be performed toprevent or reduce non-specific binding. Blocking agents for use in themethods described herein include, e.g., milk, BSA, casein, gelatin(e.g., fish gelatin), and serum (e.g., goat serum, donkey serum, horseserum, fetal bovine serum), among others.

If the detection agent is an antibody (e.g., an IAIP-specific antibodyor an IAIP ligand-specific antibody), the detection agent can bedirectly conjugated to a label (e.g., a label described above), or thedetection agent may be visualized by adding a labeled secondary antibodythat does not bind to any other antibody used in the assay (e.g., thebinding agent, if an antibody).

If the detection agent is a ligand (e.g., an IAIP ligand, such as adifferent IAIP ligand than that being detected in the assay), thedetection agent can be directly conjugated to a label (e.g., a labeldescribed above), or detected using a labeled ligand-specific antibody,which is then detected. Alternatively, the detection agent can bedetected using an unlabeled ligand-specific antibody and a labeledsecondary antibody that does not bind to any other antibody used in theassay (e.g., the binding agent, if an antibody). If the detection agentis unlabeled and additional reagents (e.g., labeled primary or secondaryantibodies) are used, one or more wash steps may be performed afterincubation with the additional reagents to minimize non-specific signal.The label can be used to measure the concentration of the IAIP-IAIPligand complex using the same substrates and imaging methods mentionedabove. The signal from the sample can be compared to signal measured insample(s) with a known concentration of the IAIP-IAIP ligand complex(e.g., to establish a standard curve). The unknown concentration in thesamples can be calculated based on an established standard curve orbased on a known reference concentration value.

If the identity of the IAIP ligand in the IAIP-IAIP ligand complex isunknown, the method can be performed using an antibody that specificallybinds to IAIP (e.g., an antibody that binds to intact IAIP and/or anIAIP heavy chain) as the binding agent. The detection step can then beperformed by adding labeled secondary antibodies specific to differentIAIP ligands, washing after incubation with the labeled secondaryantibodies, and detecting signal from the label to determine theidentity of the IAIP ligand in the IAIP-IAIP ligand complex. Labeledantibodies can be added and evaluated individually for IAIP ligandidentification, or they can be added simultaneously if different labelsare attached or conjugated to each antibody (e.g., different fluorescentdyes). Once the IAIP ligand is identified, the amount of the IAIP-IAIPligand complex in the sample can be quantified as described herein.

Quantification of IAIP Captured in the Assays

IAIP or an IAIP-IAIP ligand complex can be quantified by performing thedetection methods described herein using a sample of interest alongsidesamples containing known amounts of IAIP or an IAIP-IAIP ligand complexthat are used to create a standard curve. The sample from the subjectcan be measured at the same time as the known amounts of IAIP or anIAIP-IAIP ligand complex so that the concentration of IAIP or anIAIP-IAIP ligand complex in the sample can be determined. Theconcentration in the sample from the subject can be compared to anaverage concentration of IAIP or an IAIP-IAIP ligand complex measuredusing the same assay in a control population, such as healthy controls,to determine whether the concentration of IAIP in the sample fallswithin a normal range, or diseased controls, to determine whether theconcentration of IAIP in the sample falls within a range for the diseasestate.

IAIP or IAIP-IAIP ligand complex concentration in a sample from asubject can also be compared to IAIP or IAIP-IAIP ligand complexconcentration in a healthy control by measuring IAIP or an IAIP-IAIPligand complex in both the test sample and the control sample at thesame time using the methods described herein. Control samples includethose that are derived from the same source material (e.g., both thetest sample and the control sample are derived from the same bodilyfluid or the same tissue type). In addition to being derived from thesame source material, the test sample and control sample can also becollected from subjects of the same age and/or same sex to minimizepossible variation between subjects. If IAIP concentrations are directlycompared between a subject and a healthy control, a decrease in IAIPconcentration in the subject of 25% or more compared to the healthycontrol would indicate that the subject has or is at risk of developingan inflammatory disease or condition or an infection.

Alternatively, IAIP or IAIP-IAIP ligand complex concentration in asample from a subject can be compared to a predetermined cutoff valuefor the concentration of IAIP or IAIP-IAIP ligand complex under knownconditions (e.g., a healthy state or a disease state). The cutoff valuemay be an average concentration of IAIP or IAIP-IAIP ligand complexdetermined from a population of normal subjects or disease subjects.

Using the methods described herein, healthy control subjects have beenfound to have 400±140 μg/mL IAIP in plasma, although higherconcentrations have also been observed in healthy subjects. Subjectswith severe inflammatory disease have been found to have a meanconcentration of IAIP below about 200 μg/mL. An IAIP concentration ofabout 250 μg/mL can be used as a cut off to categorize subjects ashaving or at risk of developing a disease or condition (e.g., aninflammatory disease or condition or an infection). This categorizationcan then be used to recommend subjects for treatment or for furtherdiagnostic testing. Subjects with a moderate-to-low level of IAIP (e.g.,300 to 200 μg/mL) may benefit from repeated testing over time (e.g.,once weekly, twice monthly, once monthly, once bi-monthly, three timesannually, or biannually) to determine whether IAIP levels are constantor changing (e.g., increasing or decreasing), as these levels couldindicate risk of developing an inflammatory disease or condition or aninfection, the presence of an inflammatory disease or condition or aninfection, or they could represent the normal baseline level for asubject.

Methods of Determining Disease State or Disease Risk Using the IAIP andIAIP-IAIP Ligand Complex Detection Assays

The IAIP and IAIP-IAIP ligand complex detection methods described hereincan be used to measure IAIP and/or IAIP-IAIP ligand complexes in avariety of subjects, such as a subject having, or suspected of having, adisease or condition (e.g., an inflammatory disease or condition or aninfection (e.g., a bacterial infection)). The level of IAIP andIAIP-IAIP ligand complex in such a subject can be assessed using any ofthe above assays, e.g., for the purpose of diagnosing the presence of adisease or condition in the subject or the risk that the subject isdeveloping or will develop a disease or condition or monitoring asubject for development or resolution of a disease or condition.

Inflammatory diseases or conditions that can be, e.g., diagnosed ormonitored, using the assay methods include, e.g., acute inflammatorydisease, sepsis, septic shock, systemic inflammatory response syndrome(SIRS), trauma and/or injury (e.g., wounds, burns, lacerations,contusions, bone fractures, surgical procedures), stroke (e.g., ischemicstroke, hemorrhagic stroke), acute lung injury, acute respiratorydistress syndrome (ARDS), pneumonia (e.g., severe pneumonia, severe ornon-severe: community acquired pneumonia, hospital acquired pneumonia,nursing home acquired pneumonia), necrotizing enterocolitis, acutepancreatitis, renal diseases (e.g., acute kidney injury, liver injury,acute circulatory failure), preeclampsia, cancer, cancer metastasis,tumor invasion, peripheral artery disease, type 1 or type 2 diabetes,atherosclerotic cardiovascular disease, intermittent claudication,critical limb ischemic disease, myocardial infarction, carotidocclusion, umbilical cord occlusion, low birth-weight, premature birth,surgery-induced inflammation, abscess-associated inflammation, pulmonaryinsufficiency, peripheral neuropathy, hypoxic ischemia (e.g., neonatalhypoxic ischemic brain injury or hypoxic ischemic encephalopathy),tissue ischemia (e.g., ischemia of skeletal muscle, smooth muscle,cardiac muscle, brain, skin mesenchymal tissue, connective tissue,gastrointestinal tissue, or bone), rheumatoid arthritis, meningitis,multiple sclerosis, inflammatory bowel disease (e.g., Crohn's Disease),chronic obstructive pulmonary disease, rhinitis, preterm labor, or aninfectious disease (e.g., influenza or a viral infection, e.g., Denguefever or West Nile fever).

Infections that can be, e.g., diagnosed or monitored, using the assaymethods include, e.g., infections with gram negative bacteria, such asNeisseria species including Neisseria gonorrhoeae and Neisseriameningitidis, Branhamella species including Branhamella catarrhalis,Escherichia species including Escherichia coli, Enterobacter species,Proteus species including Proteus mirabilis, Pseudomonas speciesincluding Pseudomonas aeruginosa, Pseudomonas mallei, and Pseudomonaspseudomallei, Klebsiella species including Klebsiella pneumoniae,Salmonella species, Shigella species, Serratia species, Acinetobacterspecies; Haemophilus species including Haemophilus influenzae andHaemophilus ducreyi, Brucella species, Yersinia species includingYersinia pestis and Yersinia enterocolitica, Francisella speciesincluding Francisella tularensis, Pasturella species includingPasteurella multocida, Vibrio cholerae, Flavobacterium species,meningosepticum, Campylobacter species including Campylobacter jejuni,Bacteroides species (oral, pharyngeal) including Bacteroides fragilis,Fusobacterium species including Fusobacterium nucleatum,Calymmatobacterium granulomatis, Streptobacillus species includingStreptobacillus moniliformis, and Legionella species includingLegionella pneumophila.

The assays described herein can be used to measure IAIP levels in asubject at risk for developing an inflammatory disease or condition oran infection. Risk factors include immunosuppression, immunodeficiency(e.g., a subject that is immunocompromised), advanced age, burns (e.g.,thermal burns), trauma, surgery, foreign bodies, cancer, premature birth(e.g., a newborn born prematurely), obesity, and metabolic syndrome.

The methods described herein can be performed as part of a routinephysical examination or as a general assessment of health.

Diagnosis of Disease State or Risk of Disease

The assays described herein can be used alongside traditional diagnosticmethods to determine whether a subject has or is at risk of developingan inflammatory disease or condition or an infection. The IAIPmeasurement obtained using the assays can also be used to determinewhether a patient is a candidate for treatment with IAIP or ananti-inflammatory or anti-infective therapy or for predicting responseto administration of IAIP (e.g., a patient with low levels of IAIP canbe treated with IAIP and/or may respond favorably to treatment withIAIP). Measurement of IAIP can be followed by administration of IAIP ananti-inflammatory or anti-infective therapy to a subject, if deemedappropriate (e.g., if IAIP levels are determined to be low, e.g., atleast 25% lower than what is considered to be a normal IAIP level in ahealthy subject, or if IAIP levels are below 200 μg/mL).

Measurement of IAIP and/or an IAIP-IAIP ligand complex in a sample froma subject can be used to determine whether the subject has or is at riskof developing an inflammatory disease or condition or an infection. Themethod includes measuring the level of IAIP and/or an IAIP-IAIP ligandcomplex using one of the methods described above, and comparing thelevel to a control value (e.g., a reference sample from a healthypatient or an average value obtained from measurements of a populationof apparently healthy patients). A reduced level of IAIP in a subject ascompared to a healthy control (e.g., a level that is 25%, 30%, 40%, 50%lower or more in a subject compared to a control) or an IAIPconcentration of about 250 μg/mL or lower indicates that the subject hasor is at risk of developing an inflammatory disease or condition or aninfection. An elevated level of an IAIP-IAIP ligand complex in a subjectas compared to a healthy control (e.g., a level that is 5%, 10%, 15%,20%, 25%, 30%, 40%, 50% higher or more in a subject compared to acontrol) indicates that the subject has or is at risk of developing aninflammatory disease or infection.

The identity of the ligand bound to IAIP in the naturally occurringIAIP-IAIP ligand complex may also provide insight into the type ofinflammatory disease or infection that a subject has or is at risk ofdeveloping. For example, detecting an elevated level of an IAIP-LPScomplex in a sample from a subject may indicate that the subject has oris at risk of developing an infection (e.g., a bacterial infection), anddetecting an elevated level of an IAIP-histone complex in a sample froma subject may indicate that the subject has or is at risk of developingan acute systemic inflammatory disease (e.g., sepsis or stroke). Suchmeasurements may be used in diagnosing subjects with particularinflammatory diseases or infections, or in recommending therapies orcourses of treatment.

Disease Severity

The methods described herein can also be used to evaluate diseaseseverity. Subjects with an IAIP concentration below about 200 μg/mL, asmeasured using the assays described herein, can be categorized as havingor at high risk of developing severe inflammation or infection or ashaving a greater morbidity and/or mortality risk. Alternatively,subjects with an elevated level of an IAIP-IAIP ligand complex (e.g.,subjects with a level of an IAIP-IAIP ligand complex, such as anIAIP-LPS complex, that is 10%, 20%, 30%, 40%, 50% higher or morecompared to a healthy control or known reference concentration value) asmeasured using the assays described herein, can be categorized as havingor at high risk of developing severe inflammation or infection or ashaving a greater morbidity and/or mortality risk. Once severity isassessed, a corresponding course of treatment can be recommended.Subjects having an IAIP concentration that is indicative of severeinflammation could be selected for more frequent or more aggressivetreatment than subjects whose IAIP concentration indicates moderate orlow risk of having or developing an inflammatory disease or condition.The assays described herein can be used to measure intact IAIP, and,thus, they can be used to detect life threatening conditions and assessthe need for an appropriate therapeutic response.

Monitoring

A subject who has previously had or who is at risk of developing aninflammatory disease or condition or an infection (e.g., a subject witha genetic predisposition, a subject who has been exposed to others withthe disease or infection, or a subject having any of the risk factorsdescribed above) can be monitored using the methods described herein.Monitoring may also be a suitable approach for subjects withmoderate-to-low IAIP levels (e.g., 300 to 200 μg/mL), and/or subjectswith slightly elevated levels of an IAIP-IAIP ligand complex (e.g.,subjects with a level of an IAIP-IAIP ligand complex, such as anIAIP-LPS complex, that is 1%, 5%, or 10% higher compared to a healthycontrol or known reference concentration value), particularly if thesubjects do not present with clear symptoms of inflammation orinfection. IAIP and/or IAIP-IAIP ligand complex measurements can betaken at regular intervals (e.g., once a year, twice a year, once everythree months, once monthly, bi-monthly, or once weekly) to determinewhether IAIP and/or IAIP-IAIP ligand complex levels are constant orchanging. Increasing levels of IAIP and/or decreasing levels of anIAIP-IAIP ligand complex could indicate improvement and lead to adiscontinuation of monitoring and/or treatment. Decreasing levels ofIAIP and/or increasing levels of an IAIP-IAIP ligand complex couldindicate relapse in subjects in recovery or the development or worseningof an inflammatory disease or infection, and could lead to diagnostictesting (e.g., at a greater frequency) and the initiation of, or anincrease or change in, treatment.

Treatment Efficacy

The methods described herein can also be used to evaluate treatmentefficacy in a subject being treated (e.g., with an antibiotic,anti-inflammatory agent, anti-infective agent, or IAIP) for aninflammatory disease or condition or an infection. IAIP levels can bemeasured prior to or after the onset of treatment and then measured onan ongoing basis during treatment (e.g., once a day, once a week,bi-weekly, once a month, bi-monthly, once every three months, or twice ayear). An increase in IAIP levels (e.g., an increase of 1%, 5%, 10%,20%, 30% or more relative to a prior measurement) during the course oftreatment would indicate improvement and demonstrate the effectivenessof the treatment, while constant or decreasing IAIP levels (e.g., one ormore measurements that do not show a change or show a decrease of 1%,5%, 10%, 20%, 30% or more relative to a prior measurement) wouldindicate a lack of improvement and suggest that the course of treatmentshould be modified or changed (e.g., increased in dose or frequency orboth, changed to a different therapeutic, or modified to includeadditional therapeutic agents).

As an alternative, or in addition to measuring IAIP, detection of anIAIP-IAIP ligand complex (e.g., IAIP-LPS) can be used to evaluate theefficacy of treatment with a therapy for treating or reducing the riskof an inflammatory disease or infection. The method includes measuringthe level of the IAIP-IAIP ligand complex as a biomarker in a subjectundergoing therapy prior to or after the onset of treatment and thenmeasuring on an ongoing basis during treatment (e.g., once a day, once aweek, bi-weekly, once a month, bi-monthly, once every three months, ortwice a year). The level of the IAIP-IAIP ligand complex can be comparedthe level to a control value (e.g., a reference sample from a healthypatient or an average value obtained from measurements of a populationof control subjects (e.g., healthy patients)) or to a prior measurementtaken from the subject. A decrease in the level of the IAIP-IAIP ligandcomplex toward a “normal level” or a decrease at later time pointsduring treatment (e.g., a decrease of 1%, 5%, 10%, 20%, 30% or morerelative to a prior measurement) would indicate that the therapy isefficacious. An increase in the level of the IAIP-IAIP ligand complex(e.g., an increase of 1%, 5%, 10%, 20%, 30% or more relative to a priormeasurement or a “normal level”) would indicate that treatment isineffective and requires modification (e.g., a higher dose, morefrequent administration, or both, or a different therapeutic orcombination therapy).

Methods of Treatment

The invention also features methods of treating, preventing, or reducingthe risk of developing an inflammatory disease or condition or aninfection (e.g., a severe infection) in a subject (e.g., a human) thathas been determined to be in need according to the diagnostic methodsdescribed herein (e.g., a subject with low IAIP levels and/or elevatedlevels of an IAIP-IAIP ligand complex compared to a reference orcompared to prior measurements). The subject can be treated with astandard of care therapeutic appropriate for the disease or conditionand/or IAIP. The subject may be a neonate, a child, an adolescent, or anadult.

Prior to administration of IAIP or another therapeutic agent to asubject in need thereof, IAIP concentration can be measured in a samplefrom the subject according to the methods described herein. As analternative or in addition to measurement of IAIP levels, the method caninclude detecting an IAIP-IAIP ligand complex prior to administration ofIAIP. For example, the method can include detecting an IAIP-LPS complexin a subject having or suspected of having a bacterial infection (e.g.,a gram negative bacterial infection), and/or measuring IAIPconcentration in a sample from the subject, and administering IAIP tothe subject (e.g., administering IAIP to a subject with an increasedlevel of the IAIP-LPS complex compared to a healthy control or areference value (e.g., a level of the IAIP-LPS complex that is 1%, 5%,10%, 20%, 30% higher or more than the level in a healthy control), oradministering IAIP to a subject at risk of systemic inflammation orshock syndrome (e.g., a subject with IAIP levels at least 25% belowthose of a healthy control)).

IAIP or compositions containing IAIP can be administered to a subject inneed thereof (e.g., as determined by using one or more of the methodsdescribed herein). Subjects who can be treated with IAIP includesubjects having an infection (e.g., a gram negative bacterial infection)or subjects with an elevated risk of developing an infection (e.g.,subjects with one or more risk factors including immunosuppression,immunodeficiency (e.g., a subject that is immunocompromised), advancedage, burns (e.g., thermal burns), trauma, surgery, foreign bodies,cancer, recent birth (e.g., newborns), premature birth (e.g., newbornsborn prematurely), obesity, and metabolic syndrome). The infection canresult from endotoxins triggered by the release of lipopolysaccharide(LPS) molecules from infecting gram negative bacteria. Severe infectionby gram negative bacteria can lead to severe systemic inflammation,sepsis, shock syndrome, and death. As shown herein, IAIP binds to LPS,and, therefore, administration of IAIP can be used to treat subjectsinfected with gram negative bacteria to reduce or prevent LPS-inducedcytotoxicity.

Infections suitable for treatment with IAIP include infections with gramnegative bacteria, such as Neisseria species including Neisseriagonorrhoeae and Neisseria meningitidis, Branhamella species includingBranhamella catarrhalis, Escherichia species including Escherichia coli,Enterobacter species, Proteus species including Proteus mirabilis,Pseudomonas species including Pseudomonas aeruginosa, Pseudomonasmallei, and Pseudomonas pseudomallei, Klebsiella species includingKlebsiella pneumoniae, Salmonella species, Shigella species, Serratiaspecies, Acinetobacter species; Haemophilus species includingHaemophilus influenzae and Haemophilus ducreyi, Brucella species,Yersinia species including Yersinia pestis and Yersinia enterocolitica,Francisella species including Francisella tularensis, Pasturella speciesincluding Pasteurella multocida, Vibrio cholerae, Flavobacteriumspecies, meningosepticum, Campylobacter species including Campylobacterjejuni, Bacteroides species (oral, pharyngeal) including Bacteroidesfragilis, Fusobacterium species including Fusobacterium nucleatum,Calymmatobacterium granulomatis, Streptobacillus species includingStreptobacillus moniliformis, and Legionella species includingLegionella pneumophila.

Subjects determined to be in need of treatment (e.g., by using one ormore of the methods described herein) or who can be treated with IAIP,after such a need is determined also include subjects having or at riskof developing an inflammatory disease or condition such as acuteinflammatory disease, sepsis, septic shock, sterile sepsis, systemicinflammatory response syndrome (SIRS), trauma/injury (e.g., wounds,burns, lacerations, contusions, bone fractures, surgical procedures),stroke (e.g., ischemic stroke, hemorrhagic stroke), acute lung injury,acute respiratory distress syndrome (ARDS), pneumonia (e.g., severepneumonia, severe or non-severe: community acquired pneumonia, hospitalacquired pneumonia, nursing home acquired pneumonia), necrotizingenterocolitis, acute pancreatitis, renal diseases including acute kidneyinjury, liver injury, acute circulatory failure, preeclampsia, cancer,cancer metastasis, tumor invasion, peripheral artery disease, type 1 ortype 2 diabetes, atherosclerotic cardiovascular disease, intermittentclaudication, critical limb ischemic disease, myocardial infarction,carotid occlusion, umbilical cord occlusion, low birth-weight, prematurebirth, surgery-induced inflammation, abscess-associated inflammation,pulmonary insufficiency, peripheral neuropathy, hypoxic ischemia (e.g.,neonatal hypoxic ischemic brain injury or hypoxic ischemicencephalopathy), tissue ischemia (e.g., ischemia of skeletal muscle,smooth muscle, cardiac muscle, brain, skin mesenchymal tissue,connective tissue, gastrointestinal tissue, or bone), rheumatoidarthritis, meningitis, multiple sclerosis, inflammatory bowel disease(e.g., Crohn's Disease), chronic obstructive pulmonary disease,rhinitis, preterm labor, or an infectious disease (e.g., influenza or aviral infection, e.g., Dengue fever or West Nile fever); subjects withlow IAIP levels (e.g., IAIP levels of 250, 225, 200, 175, 150 μg/mL orlower), and subjects with increased levels of disease-associatedIAIP-IAIP ligand complexes (e.g., IAIP-LPS or IAIP-histone).

Administration

IAIPs (e.g., IαI and/or PαI), or a composition containing such proteinsand a pharmaceutically acceptable excipient, diluent, or carrier, can beadministered to a subject (e.g., a human) having or at risk ofdeveloping an inflammatory disease or condition or an infection that hasbeen determined to be in need according to the diagnostic methodsdescribed herein (e.g., a subject with low IAIP levels and/or elevatedlevels of an IAIP-IAIP ligand complex compared to a reference orcompared to prior measurements) by any suitable route, including, forexample, parenterally, by inhalation spray, topically, nasally,buccally, sublingually, intranasally, by oral administration,inhalation, suppository, rectally, vaginally, or by injection.Administration by injection includes, for example, intravenous,intraperitoneal, subcutaneous, intradermal, intracutaneous,intramuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intralesional, intravitreous, andintracranial injection. If the patient is hospitalized, the preferredmethod of administration is by intravenous injection.

The IAIPs (e.g., IαI and/or PαI) or the composition containing suchproteins may be administered to the subject one or more times every 1,2, 3, 4, 5, 6, 8, 12, or 24 hours; one or more times every 1, 2, 3, 4,5, or 6 days; or one or more times every 1, 2, 3, or 4 weeks. In othercases, the IAIPs (e.g., IαI and/or PαI) or the composition containingsuch proteins are administered as a continuous infusion.

IAIPs (e.g., IαI and/or PαI) for use in the compositions of theinvention can be obtained from, e.g., human plasma and blood by methodsknown in the art (see, e.g., U.S. Pat. No. 9,139,641, which isincorporated herein by reference in its entirety).

In particular, the IAIPs can be obtained at a purity of 80% to 100%(e.g., about 80%, about 85%, about 90%, about 95%, about 96%, about 97%,about 98%, about 99%, or about 100%) from a natural source (e.g., blood)and used to prepare a composition of the invention (see, e.g., U.S. Pat.No. 7,932,365, which is incorporated herein by reference in itsentirety). The IAIPs for use in the compositions of the invention canalso be exposed to low pH conditions (e.g., a wash buffer having a pH ofabout 4.0 or lower, e.g., about pH 3.6 or lower) during purification (asdescribed in U.S. Pat. No. 9,139,641).

The compositions may include any suitable IAIP, for example, IαI, PαI, aheavy chain, a light chain, or any combination thereof. For example, thecomposition may include IαI, PαI, and/or bikunin. In some cases, thecomposition may include IαI and PαI. The heavy chain can be H1, H2, H3,H4, or H5. The light chain can be bikunin.

The proportion or concentration of IAIPs (e.g., IαI and/or PαI) in thecompositions can vary depending upon a number of factors includingdosage, chemical characteristics (e.g., hydrophobicity), and the routeof administration. The IAIPs (e.g., IαI and/or PαI) may be present inthe composition in a physiological proportion. Physiological proportionsmay be, for example, the proportions found in a person or animal that ishealthy and/or the ratio of IαI and PαI that appears naturally in humanplasma. Physiological proportions are typically from between about 60%to about 80% IαI and between about 20% to about 40% PαI.

IAIPs (e.g., IαI and/or PαI) or compositions thereof can have ahalf-life of, for example, greater than about 1, 1.5, 2, 2.5, 3, 3.5, 4,4.5, 5, 7.5, or 10 hours. IAIPs (e.g., IαI and/or PαI) or compositionsthereof can have a half-life of greater than about 5 hours or,preferably, greater than about 10 hours. Longer half-lives arepreferred, for example, because fewer doses are required to beadministered to a subject over time.

Dosages

A pharmaceutically acceptable composition of the invention foradministration to a subject having or at risk of developing aninflammatory disease or condition or an infection that has beendetermined to be in need according to the diagnostic methods describedherein (e.g., a subject with low IAIP levels and/or elevated levels ofan IAIP-IAIP ligand complex compared to a reference or compared to priormeasurements) includes IAIPs (e.g., IαI and/or PαI) in a dosage known inthe art (see, e.g., U.S. Pat. No. 7,932,365, International PatentApplication Publication No. WO2009154695, and U.S. Patent ApplicationPublication No. 2009/0190194, each of which is incorporated herein byreference in its entirety). For example, compositions of the inventioncan be administered in a dosage ranging from about 1 mg/kg to 50 mg/kg,preferably dosages between 10 mg/kg and 30 mg/kg. The dose can beadministered one or more times every 1, 2, 3, 4, 5, 6, 8, 12, or 24hours, every 1, 2, 3, 4, 5, or 6, days, or every 1, 2, 3, or 4 weeks, oras needed. Lower or higher doses than those recited above may beadvantageous. Specific dosage and treatment regimens for any particularsubject will depend upon a variety of factors, including the activity ofthe specific composition employed, the age, body weight, general healthstatus, sex, diet, time of administration, rate of excretion, drugcombination, the severity and course of the disease (e.g., the patient'scondition and/or symptoms), the subject's disposition to the disease,and the judgment of the treating medical professional (e.g., thephysician). The IAIPs may be combined with a carrier material to producea single dosage form.

Upon improvement of the patient's condition, as evaluated based onimprovement of symptoms or by measurement of IAIP and/or an IAIP-IAIPligand complex as described herein, a maintenance dose of an IAIPcomposition or combination therapy may be administered, if necessary.Subsequently, the dosage or frequency of administration, or both, may bereduced, as a function of the reduction in symptoms, to a level at whichthe improved condition is retained. When the symptoms have beenalleviated to a desired level or IAIP has been increased and/orIAIP-IAIP ligand complexes have been decreased to a desired level,treatment may cease. Subjects may, however, require intermittenttreatment on a long-term basis upon any recurrence of disease symptomsor decrease in IAIP levels. Improvement of the condition may also bejudged based upon the level of IαIp in a biological sample derived fromthe patient (e.g., blood (e.g., whole blood, plasma, or serum),bronchial lavage fluid (BALF), sputum, urine, cerebrospinal fluid (CSF),or a tissue homogenate (e.g., a homogenate of a liver biopsy). The levelof IαIp and/or an IAIP-IAIP complex in a biological sample can bedetermined using one or more of the assays described herein.

Formulations

The invention provides methods of administering IAIP to a subject havingor at risk of developing an inflammatory disease or condition or aninfection that has been determined to be in need according to thediagnostic methods described herein (e.g., a subject found to have lowlevels of IAIP and/or elevated levels of an IAIP-IAIP ligand complex,e.g., an IAIP-LPS complex, as measured using the methods describedherein). The methods include administration of IAIPs (e.g., IαI and/orPαI), a composition that includes IAIPs (e.g., IαI and/or PαI) and apharmaceutically acceptable excipient, carrier, or diluent, or suchcompositions combined with a secondary treatment, as is describedherein. The compositions can be formulated as a solid or a liquid. Thecompositions can be formulated for administration by any suitable meansincluding those described herein.

Injectable forms of IAIPs for administration are particularly preferred.IAIPs and compositions containing the same may be formulated forintravenous, intraperitoneal, subcutaneous, intracutaneous,intramuscular, intraarticular, intraarterial, intrasynovial,intrasternal, intrathecal, intradermal, intravitreous, intralesional andintracranial injection or infusion techniques. The pharmaceuticalcompositions may be in the form of a sterile injectable preparation, forexample, as a sterile injectable aqueous or oleaginous suspension. Thissuspension may be formulated according to techniques known in the artusing suitable dispersing or wetting agents (such as, for example,TWEEN® 80) and suspending agents. The sterile injectable preparation mayalso be a sterile injectable solution or suspension in a non-toxicparenterally acceptable diluent or solvent.

The compositions may also be formulated for oral administration in anyorally acceptable dosage form including, but not limited to, capsules,tablets, pills, emulsions and aqueous suspensions, dispersions andsolutions. For preparing solid compositions, such as tablets, the IAIPsmay be mixed with a pharmaceutical excipient to form a solidpre-formulation composition containing a homogeneous mixture. This solidpre-formulation can then be subdivided into unit dosage forms of thetype described above containing from, for example, 1 mg/kg to about 50mg/kg of IAIPs (e.g., IαI and/or PαI). The solid pre-formulation cancontain about 10 mg/kg to 30 mg/kg of IAIPs (e.g., IαI and/or PαI). Thetablets or pills of the present invention can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction.

The liquid forms in which the compositions can be incorporated foradministration orally or by injection include aqueous solutions,suitably flavored syrups, aqueous or oil suspensions, and flavoredemulsions with edible oils such as cottonseed oil, sesame oil, coconutoil, or peanut oil, as well as elixirs and similar pharmaceuticalvehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedherein and/or known in the art. The compositions can be administered bythe oral or nasal respiratory route for local or systemic effect.Compositions can be nebulized by use of inert gases.

Topical administration of the compositions is useful when the desiredtreatment involves areas or organs readily accessible by topicalapplication. For application topically to the skin, the compositionshould be formulated with a suitable ointment containing the activecomponents suspended or dissolved in a carrier. Alternatively, thepharmaceutical composition can be formulated with a suitable lotion orcream containing the active composition suspended or dissolved in acarrier with suitable emulsifying agents.

The pharmaceutical compositions may also be administered in the form ofsuppositories for rectal administration. These compositions can beprepared by mixing a composition of this invention with a suitablenon-irritating excipient which is solid at room temperature but liquidat the rectal temperature and therefore will melt in the rectum torelease the active components. Topically-transdermal patches are alsoincluded in this invention.

The compositions administered to a subject can be in the form of one ormore of the pharmaceutical compositions described above. Thesecompositions can be sterilized by conventional sterilization techniquesor may be sterile filtered. Aqueous solutions can be packaged for use asis, or lyophilized, the lyophilized preparation being combined with asterile aqueous carrier prior to administration.

Other delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of compositions of the invention, increasing convenienceto the subject and the physician. Many types of release delivery systemsare available and known to those of ordinary skill in the art. Theyinclude polymer base systems such as those described in (U.S. Pat. No.3,773,919; European Patent No. 58,481, European Patent No. 133, 988,Sidman, K. R. et al., Biopolymers 22: 547-556, and Langer, R. et al., J.Biomed. Mater. Res. 15:267-277; Langer, R. Chem. Tech. 12:98-105). Otherexamples of sustained-release compositions include semi-permeablepolymer matrices in the form of shaped articles, e.g., films, ormicrocapsules. Delivery systems also include non-polymer systems thatare: lipids; hydrogel release systems; sylastic systems; peptide basedsystems; wax coatings; compressed tablets using conventional binders andexcipients; partially fused implants; and the like. Methods forpreparation of such formulations will be apparent to those skilled inthe art (see, e.g., U.S. Pat. Nos. 4,452,775, 4,667,014, 4,748,034 and5,239,660, 3,832,253, and 3,854,480).

Methods of formulating pharmaceutical agents are known in the art, e.g.,Niazi, Handbook of Pharmaceutical Manufacturing Formulations (SecondEdition), CRC Press 2009, describes formulation development for liquid,sterile, compressed, semi-compressed and OTC forms. Transdermal andmucosal delivery, lymphatic system delivery, nanoparticles, controlleddrug release systems, theranostics, protein and peptide drugs, andbiologics delivery are described in Wang et al., Drug Delivery:Principles and Applications (Second Edition), Wiley 2016; formulationand delivery of peptide and protein agents is described, e.g., in Banga,Therapeutic Peptides and Proteins: Formulation, Processing, and DeliverySystems (Third Edition), CRC Press 2015.

Combination Therapies

The methods of the invention also include administering orco-administering a second treatment (e.g., as a standalone therapy or inaddition to IAIPs (e.g., IαI and/or PαI) or a composition thereof) forthe treatment of an inflammatory disease or condition (e.g., sepsis,septic shock, sterile sepsis, SIRS, trauma/injury (e.g., wounds, burns,lacerations, contusions, bone fractures, surgical procedures), stroke(e.g., ischemic stroke, hemorrhagic stroke), acute lung injury, ARDS,pneumonia (e.g., severe pneumonia, severe or non-severe: communityacquired pneumonia, hospital acquired pneumonia, nursing home acquiredpneumonia), necrotizing enterocolitis, acute pancreatitis, renaldiseases including acute kidney injury, liver injury, acute circulatoryfailure, preeclampsia, cancer, cancer metastasis, tumor invasion,peripheral artery disease, type 1 or type 2 diabetes, atheroscleroticcardiovascular disease, intermittent claudication, critical limbischemic disease, myocardial infarction, carotid occlusion, umbilicalcord occlusion, low birth-weight, premature birth, surgery-inducedinflammation, abscess-associated inflammation, pulmonary insufficiency,peripheral neuropathy, hypoxic ischemia, tissue ischemia, rheumatoidarthritis, meningitis, multiple sclerosis, inflammatory bowel disease(e.g., Crohn's Disease), chronic obstructive pulmonary disease,rhinitis, preterm labor, or an infectious disease) or an infection(e.g., a bacterial infection). For example, the second treatment mayinclude administering an antibiotic agent if the subject has or is atrisk of developing an bacterial infection, an antiviral agent if thesubject has or is at risk of developing an viral infection (e.g., Denguefever or West Nile fever), an antifungal agent if the subject has or isat risk of developing a fungal infection, an antiparasitic agent if thesubject has or is at risk of developing a parasitic infection, ananti-inflammatory agent if the subject has or is at risk of developingan inflammatory disease or condition described herein, an anti-canceragent if the subject has or is at risk of developing cancer or cancermetastasis, an anti-coagulant if the subject has or is at risk of strokeor myocardial infarction, an immunomodulatory agent if the subject hascancer or an autoimmune disease or condition (e.g., inflammatory boweldisease or rheumatoid arthritis), and a bronchodilator agent, acomplement inhibitor, a vasopressor, a sedative, or mechanicalventilation if the subject has or is at risk of developing acute lunginjury, ARDS, or pneumonia.

When the method includes administering a combination of IAIPs (e.g., IαIand/or PαI), or a composition including IAIPs (e.g., IαI and/or PαI) anda pharmaceutically acceptable excipient, diluent, or carrier, and one ormore second treatment agents, each agent is present at a dosage level ofbetween about 1 to 100%, and more preferably between about 5 to 95%, ofthe dosage normally administered in a monotherapy regimen. The agent(s)of the second treatment may be administered separately, as part of amultiple dose regimen, from the IAIPs (e.g., IαI and/or PαI) or thecomposition thereof. The IAIPs and agent(s) of the second treatment canbe administered simultaneously or sequentially in any order.Alternatively, the agent(s) of the second treatment may be part of asingle dosage form, e.g., mixed together with the IAIPs (e.g., IαIand/or PαI) in a single composition.

Agents that can be administered in combination with IAIPs (e.g., IαIand/or PαI) include dideoxynucleosides, e.g. zidovudine (AZT),2′,3′-dideoxyinosine (ddl) and 2′,3′-dideoxycytidine (ddC), lamivudine(3TC), stavudine (d4T), and TRIZIVIR (abacavir+zidovudine+lamivudine);non-nucleosides, e.g., efavirenz (DMP-266, DuPontPharmaceuticals/Bristol Myers Squibb), nevirapine (BoehringerIngleheim), and delaviridine (Pharmacia-Upjohn); TAT antagonists such asRo 3-3335 and Ro 24-7429; protease inhibitors, e.g., furin inhibitors,indinavir (Merck), ritonavir (Abbott), saquinavir (Hoffmann LaRoche),nelfinavir (Agouron Pharmaceuticals), 141 W94 (Glaxo-Wellcome),atazanavir (Bristol Myers Squibb), amprenavir (GlaxoSmithKline),fosamprenavir (GlaxoSmithKline), tipranavir (Boehringer Ingleheim),KALETRA (lopinavir+ritonavir, Abbott), and other agents such as9-(2-hydroxyethoxymethyl)guanine (acyclovir); interferon, e.g.,alpha-interferon, interleukin II, and phosphonoformate (Foscarnet); orentry inhibitors, e.g., T20 (enfuvirtide, Roche/Trimeris) or UK-427,857(Pfizer), levamisol or thymosin, cisplatin, carboplatin, docetaxel,paclitaxel, fluorouracil, capecitabine, gemcitabine, irinotecan,topotecan, etoposide, mitomycin, gefitinib, vincristine, vinblastine,doxorubicin, cyclophosphamide, celecoxib, rofecoxib, valdecoxib,ibuprofen, naproxen, ketoprofen, dexamethasone, prednisone,prednisolone, hydrocortisone, acetaminophen, misonidazole, amifostine,tamsulosin, phenazopyridine, ondansetron, granisetron, alosetron,palonosetron, promethazine, prochlorperazine, trimethobenzamide,aprepitant, diphenoxylate with atropine, and/or loperamide; andanti-coagulants, e.g., Anti-thrombin III and activated Protein C.

Additional exemplary agents that can be administered in combination withIAIPs (e.g., IαI and/or PαI) or compositions thereof are discussedbelow.

Antibiotic Agents

If the subject has or is at risk of developing a bacterial infection(e.g., necrotizing enterocolitis or a gram negative bacterial infection)the second treatment may include an antibiotic agent that is used totreat a bacterial infection. Non-limiting examples of antibiotic agentsinclude amoxicillin, penicillin, doxycycline, clarithromycin,benzylpenicillin, azithromycin, daptomycin, linezolid, levofloxacin,moxifloxacin, gatifloxcin, gentamicin, macrolides, cephalosporins,azithromycin, ciprofloxacin, cefuroxime, amoxillin-potassiumclavulanate, erythromycin, sulfamethoxazole-trimethoprim, doxycyclinemonohydrate, cefepime, ampicillin, cefpodoxime, ceftriaxone, cefazolin,erythromycin ethylsuccinate, meropenem, piperacillin-tazobactam,amikacin, erythromycin stearate, cefepime in dextrose, doxycyclinehyclate, ampicillin-sulbactam, ceftazidime, gemifloxacin, gentamicinsulfate, erythromycin lactobionate, imipenem-cilastatin, cefoxitin,cefditoren pivoxil, ertapenem, doxycycline-benzoyl peroxide,ampicillin-sulbactam, meropenem, cefuroxime, cefotetan,piperacillin-tazobactam, broad-spectrum fluoroquinolones (which may beused, for example, to treat pneumonia caused by atypical pathogens suchas Mycoplasma pneumoniae or Chlamydophila pneumoniae), and others knownin the art.

Antiviral Agents

If the subject has or is at risk of developing a viral infection (e.g.,Dengue fever or West Nile fever), the second treatment may include anantiviral agent that is used to treat a viral infection. Non-limitingexamples of antiviral agents include zanamivir, oseltamivir, permivir,ribavirin, acyclovir, ganciclovir, foscarnet, cidofovir, and othersknown in the art.

Antifungal Agents

If the subject has or is at risk of developing a fungal infection, thesecond treatment may include an antifungal agent that is used to treat afungal infection. Non-limiting examples of antifungal agents includeamphotericin, caspofungin, voriconazole, itraconazole, posaconazole,fluconazole, flucytosine, and others known in the art.

Antiparasitic Agents

If the subject has or is at risk of developing a parasitic infection,the second treatment may include an antiparasitic agent that is used totreat a parasitic infection (e.g., a parasitic protozoan infection.Non-limiting examples of antiparasitic agents include nitazoxanide,melarsoprol, eflornithine, metronidazole, tinidazole, miltefosine,mebendazole, pyrantel pamoate, thiabendazole, diethylcarbamazine,ivermectin, albendazole, praziquantel, rifampin, and others known in theart.

Anti-Inflammatory Agents

If the subject has or is at risk of developing an inflammatory diseaseor condition described herein, the second treatment may include ananti-inflammatory agent that is used to treat or reduce inflammation.Non-limiting examples of anti-inflammatory agents includecorticosteroids, statins, steroids, nonsteroidal anti-inflammatorydrugs, glucocorticoids, and others known the art.

Bronchodilators

If the subject has or is at risk of developing acute lung injury, ARDS,or pneumonia, the second treatment may include a bronchodilator that isused to relax the bronchial muscles allowing airways to be larger andair to pass through the lungs. Non-limiting examples of bronchodilatorsinclude beta 2 agonists, xanthines, ipratropium, oxitropium, muscarinicreceptor antagonists, ipratropium, oxitropium, theophylline,theobromine, caffeine, salbutamol, isoproterenol, albuterol,levalburerol, pirbuterol, metaproterenol, terbutaline, salmeterol,formoterol, and others known in the art.

Vasopressors

If the subject has or is at risk of developing acute lung injury, ARDS,pneumonia, or trauma/injury (e.g., wounds, burns, or surgicalprocedures), the second treatment may include a vasopressor that causesvasoconstriction and/or an increase in blood pressure. Non-limitingexamples of vasopressors include epinephrine, isoproterenol,phenylephrine, norepinephrine, dobutamine, ephedrine, droxidopa, andothers known in the art.

Sedatives

The second treatment may include a sedative. Non-limiting examples ofsedatives include propofol, diprivan, morphine, fentanyl, midazolam,lorazepam, precede, infumorph, dexmedetomidine, alfentanil, and othersknown in the art.

Complement Inhibitors

If the subject has or is at risk of developing acute lung injury, ARDS,or pneumonia, the second treatment may include an inhibitor ofcomplement activation. The composition may inhibit activation of one ormore complement components such as C1, C2, C3 (e.g., C3a and C3b), C4(e.g., C4b), C5 (e.g., C5a and C5b), C6, C7, C8, C9, membrane attackcomplex, Factor B, Factor D, MASP-1, and MASP-2, or fragments thereof.The complement inhibitors may include protease inhibitors such as C1-INHand Rhucin/rhC11 NH, soluble complement regulators such as sCR1/TP10,CAB-2/MLN-2222, therapeutic antibodies such as eculizumab/SOLIRIS®,Pexelizumna, ofatumumab, complement component inhibitors such ascompstatin, receptor antagonists such as PMX-53 and rhMBL.

Kits

The invention also features kits for use in measuring IAIP in a sample(e.g., a fluid sample) from a patient (e.g., a human patient, such as aneonate, a child, an adolescent, or an adult). The kit may include oneor more of the following: a support (e.g., a plate (e.g., a multi-wellplate)), particles (e.g., magnetic particles, e.g., nanoparticles,magnetic nanoparticles), biochips, resins, containers (e.g., tubes),membranes (e.g., nitrocellulose membranes, PVDF membranes), test strips(e.g., cellulose, glass fiber, or nitrocellulose) or beads (e.g.,protein A or protein G beads, magnetic beads, glass beads, plasticbeads)) containing an immobilized IAIP binding agent (e.g., anIAIP-specific antibody or an IAIP ligand), a labeled IAIP detectionagent (e.g., an IAIP ligand or IAIP-specific antibody), a wash buffer, ablocking agent, a substrate for detection of the label, a dilutionagent, and instructions for performing the detection assay. The bindingagent and detection agent may be provided in containers, or the bindingagent may be provided pre-attached to the support (e.g., the bindingagent is already attached to the plate or test strip).

The invention also features kits for use in measuring an IAIP-IAIPligand complex in a sample (e.g., a fluid sample) from a patient. Thekit may include one or more of the following: a support (e.g., a plate(e.g., a multi-well plate)), particles (e.g., magnetic particles, e.g.,nanoparticles, magnetic nanoparticles), biochips, resins, containers(e.g., tubes), membranes (e.g., nitrocellulose membranes, PVDFmembranes), test strips (e.g., cellulose, glass fiber, ornitrocellulose) or beads (e.g., protein A or protein G beads, magneticbeads, glass beads, plastic beads)) containing an immobilized bindingagent (e.g., an IAIP-specific antibody, a different IAIP ligand, or anantibody that binds specifically to the IAIP ligand), a labeleddetection agent (e.g., a different IAIP ligand, an IAIP-specificantibody, or an antibody that binds specifically to the IAIP ligand),wash buffers, a blocking agent, a substrate for detection of the label,a dilatation agent, and instructions for performing the detection assay.The binding agent and detection agent may be provided in containers, orthe binding agent may be provided pre-attached to a support (e.g., thebinding agent may be already attached to a plate or test strip).

EXAMPLES

The following examples are provided to further illustrate someembodiments of the present invention, but are not intended to limit thescope of the invention; it will be understood by their exemplary naturethat other procedures, methodologies, or techniques known to thoseskilled in the art may alternatively be used.

Example 1: Heparin-IAIP Assay

Preparation of Biotinylated Heparin

Heparin (Heparin Sodium Injection USP, Sagent Pharmaceuticals, Cat # NDC25021-400-30) was conjugated with biotin using the Biotin Hydrazidereagent (ApExBIO, Cat # A87007) according to the manufacturer'sinstructions. Briefly, 1000 IU heparin solution was mixed with 0.25 mgcrosslinker reagent EDC (1-(3-Dimethylaminopropyl)-3-3ethylcarbodiimidehydrochloride, Alfa Aesar Cat # A10807) and 0.5 mM Biotin hydrazide thathad been previously dissolved in DMSO in 0.1 M MES buffer pH 4.7 withgentle mixing at room temperature for 3 hrs. The unconjugated biotin andbuffer exchange was carried out by ultrafiltration on an Amicon Ultracentrifugal filter device with 5 kDa cut off filter membrane(Millipore). Following dilution in d-H2O, the biotinylated Heparin wasready for use in the assay.

“Sandwich Type” Heparin-IAIP ELISA

Purified mouse monoclonal antibody against the light chain of human IAIP(MAb 69.26) was immobilized on a 96-well microplate (Immulon 600,Greiner BioOne) at 200 ng/well at 32° C. for 2 hrs. After blocking with5% non-fat dried milk for 1 hr and washing with TBS-T (TBS+0.05% Tween20), unknown samples and known IAIP standard were diluted in TBS+0.1%Tween 20 and added to the microplate (final volume 50 uL/well). Thesamples and the serially diluted IAIP standard solution were incubatedfor 1 hr at 32° C. After several washes of the microplate with TBS-T,biotinylated heparin was diluted in a buffer containing 20 mM Aceticacid+25 mM NaCl, pH 4.0 (1:2500) and 50 μL was added per well. Thebiotinylated heparin was incubated for 30 minutes at 32° C. and themicroplate was then washed at least three times using TBS-T. Finally,HRP-conjugated Streptavidin (Pierce) diluted at 1:5000 (50 uL/well) wasadded to the microplate. Following washing, 50 μL of the substrate TMBwas added (Neogen Enhanced K-Blue TMB substrate) and the reaction wasstopped with the addition of 50 μL 1 M HCl and the color change was readon spectrophotometer (Molecular Devices) at 450 nm wavelength. Thestandard curve was generated using four-point logistic regression(SoftMax Pro software, Molecular Devices) and a seven-point curve wasplotted from maximum IAIP concentration of 2.0 pg/mL to 0.03125 pg/mLwith serial two-fold dilution as shown in FIG. 3B. The IAIPconcentration of the unknown samples was calculated based on thegenerated standard curve.

Example 2: LPS-IAIP Assay

Preparation of Biotinylated Endotoxin/LPS (Lipopolysaccharide):

Lipopolysaccharide (LPS/endotoxin) from Escherichia coli 055:B5 (SigmaCatalog # L2280) was labeled with biotin using Biotin Hydrazide reagent(ApExBIO, Cat # A87007) according to the manufacturer's instructions andsimilarly to the protocol used for heparin. 10 mg LPS was reconstitutedin 0.1 M MES buffer and 2.5 mM Biotin-Hydrazide and 2.5 mg EDC(1-(3-Dimethylaminopropyl)-3-3ethylcarbodiimide hydrochloride, AlfaAesar Cat # A10807) were gently mixed for 3 hrs at room temperature. Theremoval of unconjugated LPS and buffer exchange were carried out byultrafiltration on Amicon Ultra centrifugal filter device with a 5 kDacut off filter membrane (Millipore). Following dilution in d-H2O, thebiotinylated LPS was ready for use in the assay.

“Sandwich Type” LPS-IAIP ELISA

Similar to the heparin-IAIP protocol described above, purified mousemonoclonal antibody against the light chain of human IAIP (MAb 69.26)was immobilized on 96-well microplate (Immulon 600, Greiner BioOne) at50 ng/well at 32° C. for 2 hrs. After blocking with 5% non-fat driedmilk for 1 hr and washing with TBS-T (TBS+0.05% Tween 20), unknownsamples and known IAIP standard were diluted in TBS+0.1% Tween 20 andadded to the microplate (final volume 50 uL/well). The samples and theserially diluted IAIP standard solution were incubated for 1 hr at 32°C. After several washes of the microplate with TBS-T, biotinylated LPSwas diluted in a buffer containing 20 mM Acetic acid+25 mM NaCl, pH 4.0(1:32,000) and 50 uL was added per well. The biotinylated LPS wasincubated for 30 minutes at 32° C. and the microplate was then washed atleast three times using TBS-T. Finally, HRP-conjugated Streptavidin(Pierce) diluted at 1:10,000 (50 μL/well) was added to the microplate.Following washing, 50 μL of the substrate TMB was added (Neogen EnhancedK-Blue TMB substrate) and the reaction was stopped with the addition of50 μL 1 M HCl and the color change was read on spectrophotometer(Molecular Devices) at 450 nm wavelength. The standard curve wasgenerated using four-point logistic regression (SoftMax Pro software,Molecular Devices) and a seven-point curve was plotted from maximum IAIPconcentration of 2.0 μg/mL to 0.03125 μg/mL with serial two-folddilution as shown in FIG. 3C.

Example 3: Analysis of Blood Samples from Patients Diagnosed with SevereCommunity Acquired Pneumonia (sCAP)

Serial blood samples were collected from patients with a confirmeddiagnosis of sCAP who were hospitalized in the Intensive Care Unit atRhode Island Hospital. 16 patients were enrolled in the study and plasmawas collected on days 0 (time of admission to the ICU), 1, 3 and 7. Thelevel of IAIP was determined using the established competitive ELISA(FIG. 2A) and both sandwich-type ELISAs using biotinylated heparin orbiotinylated LPS as detecting molecules (FIG. 3A). Blood samples from 95healthy controls aged between 17 to 71 years old (obtained from healthyblood donors and purchased from Rhode Island Blood Center) were includedin this study to compare IAIP levels in health controls to the levelsmeasured in sCAP patients. The results are shown in FIGS. 4, 5, and 6.

The results indicate that IAIP levels were significantly lower in sCAPpatients compared to healthy controls at the time of hospitalization andduring disease progression (up to 7 days). Therefore, IAIP levels can beused to guide physicians in evaluating prognosis and making therapeuticdecisions. Although the competitive ELISA showed decreased IAIP levelsin sCAP patients, the “sandwich-type” ELISA assays usingbiotin-conjugated heparin and/or LPS as specific binding ligands yieldedresults that were more statistically significant (p value=0.0001 betweenIAIP level at day 0,1,3 and 7 compared to healthy controls). The“sandwich-type” ELISA assays using biotin-conjugated heparin and/or LPSalso yielded a lower concentration of IAIP in sCAP patients, giving riseto a greater difference between IAIP levels measured in patients withsCAP and healthy controls. These data indicate that the “sandwich-type”ELISA assays using labeled IAIP ligands have increased sensitivity andpotentially greater accuracy than competitive ELISA assays.

The same assays were also used to evaluate samples from subjects withsepsis that were admitted to the intensive care unit at Rhode IslandHospital. These assays yielded similar results. While the IAIP values ofnormal healthy controls are similar in the competitive ELISA(mean±SEM=328.9+6.34 μg/mL) and both the LPS or heparin-IAIP assay(337.7±9.05 and 421.8±14.47 μg/mL), the levels of IAIP in pneumonia andseptic groups were significantly higher in the competitive ELISA, whichresulted in statistically less significant differences or no differencebetween the diseased groups and healthy controls (FIGS. 8A-8C).

Example 4: Analysis of the Binding of Heparin to IAIP

The evaluation of the specific binding of heparin to IAIP was carriedout using western blot analysis. Highly purified IAIP (1 μg), normalhuman plasma (1 μL), purified bikunin (Ulinastatin for Injection,Techpool, 2 μg) and human serum albumin (HSA, 2 μg) as a negativecontrol were separated on a 7.5% SDS-PAGE gel and transferred onto anitrocellulose membrane. Following blocking with 5% non-fat dried milk,the nitrocellulose membrane was incubated with biotin-conjugated heparin(1:500 in TBS) overnight at room temperature. After several washes withTBS+0.05% Tween, HRP-conjugated streptavidin (1:15,000) was added andincubated for 1 hr at room temperature. Following washing, the substrateMetal-enhanced DAB (Pierce) was added to visualize the reactive bands.

The biotin-conjugated heparin bound specifically to purified IAIP (250kDa IαI and 125 kDa PαI) as shown in Lane 2 (FIG. 7), similar to MAb69.26 (monoclonal antibody against human IAIP). In contrast to MAb 69.26that bound to the light chain (bikunin) (lane 3), biotin-conjugatedheparin did not bind to the light chain of IAIP, suggesting that heparinbinds to IAIP via the heavy chain. The heavy chains of IAIP appear to bebound specifically to heparin.

Multiple reactive protein bands were detected by biotin-conjugatedheparin in human plasma (FIG. 7, Lane 1) and binding to HSA was notdetectable.

Moreover, free bikunin was not detected in the heparin-IAIP or LPS-IAIPELISA, suggesting that only IAIP complexes with the intact heavy chainare measured using the heparin or LPS-IAIP ELISA. These data indicatethat the sandwich-type ELISA using heparin and/or LPS (or other IAIPligands) provides an assessment of the circulating IAIP complex in asubject (e.g., a subject with a pathological condition) that hasincreased accuracy and robustness relative to other known IAIP assays,such as the competitive IAIP assay (FIG. 2A).

Example 5: Binding of Biotinylated LPS to Immobilized Plasma-DerivedIAIP

To investigate the direct binding of LPS to IAIP in vitro, IAIP andother control proteins (BSA=bovine serum albumin; bikunin (the lightchain of IAIP); and purified IgG of monoclonal antibody MAb 69.26) at1000 μg/well were immobilized on a 96-well microplate (Greiner Microlon600) and blocked with non-fat dried milk (5% in TBS+0.1% Tween 20).Following washing, 100 μg biotinylated LPS (Lipopolysaccharides fromEscherichia coli 055:B5, purchased from Sigma, Cat # L2880) was added toeach well and incubated in TBS+150 mM CaCl2) buffer for 1 hr at roomtemperature. Following several washes with TBS+0.1% Tween,HRP-conjugated Streptavidin was added and incubated for 1 hr. Finally,TMB substrate was added and the reaction was stopped by adding 1 M HClsolution. The color change and absorbance were measuredspectrometrically at 450 nm. Significant binding of biotinylated LPSmolecules to IAIP was found compared to BSA, which served as a positivecontrol, as it has been described to bind LPS (David S A, et al., InnateImmunity 1995; 2(2):99-106), or IgG of MAb 69.26 (negative control)(FIG. 9). LPS did not bind significantly to the light chain of IAIP(bikunin) suggesting that the heavy chain of IAIP facilitates thebinding to LPS. The binding might also be mediated by theglycosaminoglycan chains.

Example 6: Binding of Biotinylated IAIP to Immobilized LPS

In a reversal of the previous experiment, biotinylated IAIP (4 μg/well)was added and incubated for 1 hr to immobilized LPS (100 μg/well), BSA(2 μg/well), IgG of MAb 69.26 (2 μg/well) as a positive control, andnon-fat dried milk (2 μg/well) as negative control (blank). Followingseveral washes, HRP-conjugated streptavidin was added and subsequentlyTMB substrate was added to the microplate wells. Significant binding ofbiotinylated IAIP to immobilized LPS was detected, while significantlylower binding of IAIP to immobilized BSA or non-fat dried milk wasobserved FIG. 10). IgG of MAb 69.26 (monoclonal antibody specific tohuman IAIP) that served as a positive control in this experiment boundstrongly to biotinylated IAIP.

Example 7: Analysis of pH Effects on IAIP-LPS Binding

To further characterize the binding of IAIP and LPS, similar solid phasebinding experiments were carried out. Biotinylated IAIP was incubatedwith immobilized LPS, IgG of MAb 69.26 (positive control), or BSA(negative control) under various pH conditions. Acetate buffer (50 mM)was used for the low pH solution (pH 3-6) and Tris-HCl buffer (50 mM)was used to obtain a neutral or higher pH solution (pH 7-9). Whilebiotinylated IAIP bound strongly at pH 5, the binding was not observedat pH 3 or 4 (FIG. 11). Decreased binding of IAIP was observed when pHwas increased above pH 5. The binding of IAIP to its specific monoclonalantibody MAb 69.26 peaked at pH 7 but, interestingly, did notsignificantly change when pH was increased up to pH 9. Similarly, at pH3 and 4, IAIP binding to the monoclonal antibody was negligible. Therewas no binding observed between biotinylated IAIP and BSA. The resultsclearly suggest that the optimum binding between IAIP and LPS occurs ata range of pH 4-7, and in particular, at about pH 5.

Example 8: Analysis of the Effect of Salt Concentration on IAIP-LPSBinding

The effect of salt concentration was investigated by adding increasingamounts of salt (NaCl) to the buffer during the incubation ofbiotinylated IAIP with the immobilized LPS on the microplate. Thebinding of IAIP to LPS decreased with increasing salt concentration,however the decrease was not significant even at a salt concentration of1200 mM, suggesting that the binding of IAIP to LPS was relativelystrong and specific, similar to the specific binding of IAIP to themonoclonal antibody (MAb 69.26) against human IAIP which was used as thepositive control in this experiment (FIG. 12).

Example 9: Analysis of the Effect of Non-Ionic Detergents (NP-40 andTween-20) on IAIP-LPS Binding

Further investigation was carried out to study the effects of increasingconcentrations of non-ionic detergents nonyl phenoxypolyethoxylethanol(NP-40) (FIG. 13) and Tween 20 (FIG. 14) on the binding of biotinylatedIAIP to immobilized LPS. Increased binding was observed when 0.05% (w/w)NP-40 or Tween-20 was added to the binding reaction of IAIP to LPS,compared to TBS buffer alone without the addition of detergent. The lowamount of detergent might facilitate the binding to LPS, suggesting thatthe lipid binding domain of the LPS molecule might be involved in thisinteraction. Even when the amount of the detergent was increased up to1%, the binding of IAIP was still relatively strong as most of the IAIPwas still bound to LPS indicating a strong interaction between IAIP andLPS in vitro. Similarly, biotinylated IAIP strongly bound to MAb 69.26with high specificity and affinity and the addition of detergent up to1% did not significantly change the binding. In contrast, the negativecontrol (BSA) did not show any binding to IAIP.

Example 10: Quantification of IAIP Using a Rapid Lateral-FlowImmunoassay (LFIA)

The methods described herein can be used for the rapid quantification ofIAIP using a lateral-flow immunoassay (LFIA). The “sandwich type” IAIPELISA described and used in Examples 1-3 can be adapted to LFIA, apoint-of-care (POC), rapid, reliable, quantitative and user-friendlytest that can be used to identify high-risk subjects (e.g., infants,adolescents, or adults) with life threatening, severe inflammatoryconditions (e.g., neonatal sepsis (NS) and necrotizing enterocolitis(NEC)). The LFIA can a) measure a linear range between 20 and 700 μg/mlIAIP; b) exhibit high precision (e.g., variability or error of <5%) forIAIP levels near 150 μg/ml (e.g., about 100 to about 200 μg/mL); c)obtain results in an hour or less (e.g., 15 minutes or less, such asless than 10, 7, or 5 minutes or less); and d) require a small samplevolume<150 μL of plasma or whole blood samples (e.g., 150, 100, 75, 50,25, 15, μL or less).

The test can be used to quickly identify a dysregulated inflammatoryresponse in subjects (e.g., preterm newborns, infants, adolescents oradults). It is known that early intervention is important for improvingsurvival in patients experiencing an inflammatory disease or conditionor an infection.

In newborns, for example, the ability to apply early interventionprotocols is often limited by the difficulty in identifying suchconditions (e.g., NS and NEC) from other less serious diseases. Theexcellent negative predictive value of IAIP can help guide clinicians tomake difficult decisions, such as, e.g., early termination of antibiotictreatment in a subject (e.g., an infant, an adolescent, or an adult) inwhom current tests are uninformative. Furthermore, as therapeuticproteins, IAIP might be beneficial in critically ill subjects (e.g.,infants, adolescents, or adults) and the blood levels can serve as auseful theranostic marker to help physicians make informed decisionsabout a replacement therapy with IAIP or other adjunctive therapeuticagents and to monitor disease progression.

A rapid point-of care test based on IAIP that can be used to influencetherapeutic decisions, such as initiation and duration of treatment(e.g., antibiotic treatment), using a simple, user-friendly and portabledevice with accurate quantitative results readable within a short timeperiod (e.g., 15 min. or less) is not only innovative, but alsoclinically useful to help reduce morbidity and mortality.

The LFIA involves adding a sample from a subject to a test strip (e.g.,cellulose, glass fiber, or nitrocellulose), which is then pushed intothe strip using a buffer. The strip contains an IAIP binding agent(e.g., an IAIP-specific antibody or an IAIP ligand). The labeleddetection agent (e.g., an IAIP-specific antibody or an IAIP ligand) canbe added to the strip before, after, or concurrently with the sample,and visualized using standard methods for the rapid quantification ofIAIP.

a) Gold-Conjugated Reagents:

Gold nanoparticles can be used to label the reagents in the (LFIA)format due to its superior colloidal stability. A stable formulation ofgold nanoparticles that are functionalized with hydrazides arecommercially available (Innova Bioscience). While usually antibodies arecovalently attached to colloidal gold as detecting reagents, heparin orLPS can be similarly conjugated via their sugar chains using theInnovaCoat Gold nanoparticles. The gold conjugation of LPS and heparincan be performed in a small scale and tested for their stability andperformance in the LFIA.

b) Optimization of Capture, Detection, Sample Reagents and Conditions ofthe LFIA:

Building upon the experience with the competitive LFIA developmentdescribed below, the formulation and coating conditions of MAb 69.26 onthe capture line and other factors, such as blocking agents,surfactants, and carbohydrates, can be selected to reduce non-specificbinding and to improve flow characteristics. The optimal dilution factorand suitable diluent that reduces interference and non-specific bindingcan also be determined. Multi-factorial design of experiments (DoE) canbe used to determine the interaction of the following parameters: 1)binding agent concentration, 2) coating pH and buffer type (range=4-10),3) coating duration and 4) type of blocking agent (e.g., bovine serumalbumin, fish gelatin, etc.).

c) Test Strip Components, Housing and Reader:

The test strip can be housed in a plastic cassette with sample well andtest read windows placed over the appropriate areas of the strip. Avariety of standardized strip dimensional configurations can be used. Asuitable housing can be chosen, such as one that is adaptable to theDetekt or ESEQuant reader. The composition of sample and conjugate pad,as well as the pore size of the nitrocellulose membrane, can be selectedto achieve a desired rate of capillary action, and hence, reaction timeof the sample with detector reagent.

LFIA performance goals can be optimized by changing, e.g., theformulations and processes for the striping conditions of the Test linereagent, the conjugation method of the detecting molecules (e.g., LPS orheparin), and the titration of the detection agents, as well as theoptimal material and dimensions of pads and wicks, as described below:

a. Optimization of Test and Control Lines.

Membrane stripping conditions can be optimized for both Test Line (MAb69.26) and Control Line (LPS-binding proteins or heparin-bindingproteins (for e.g. clotting factor IX) of the test strip. A 30 minute at37° C. drying period can be used to immobilize the Test and ControlLines, as robust manufacturing procedures for LFIA membranes.Multi-factorial DoE can be used to determine the interaction of thefollowing membrane stripping parameters: 1) binding agent concentration,2) pH and buffer type (range=pH 4-10), 3) salt concentration and 4)striping volume (1-3 μL/strip) and inkjet speed. Incorporating membranestriping improvements from initial optimization studies. Multi-factorialDoE can also be used to determine the efficacy of membrane co-coatingwith various concentrations (0-1 μg/strip) of protein blockers,surfactants, carbohydrates, and other agents which may reduce goldparticle non-specific binding or improve flow characteristics of themembrane. Based on our solid phase binding studies, LPS and heparin bindsignificantly stronger to IAIP at pH lower than 5. Similar conditionscan be used in the LFIA.

b. Optimization of Test Strip Parameters.

The composition of the sample pad and conjugate pad, as well as the poresize of the nitrocellulose membrane, can affect the rate of capillaryaction, and hence, reaction time of the sample with gold detectorreagent, and thus, can be selected to achieve desired results. We havedetermined that a fast flow NC (HF090, Millipore) met designspecifications for sensitivity and time to result. DoE can be used toassess types of filter, and pad and wick materials for the assay.Similarly, the size and geometry of the sample pad and conjugate pad canbe adjusted to achieve desired results.

The sample pad can be optimized to: 1) accept undiluted sample, 2)provide the necessary formula to minimize non-specific binding and 3)accept whole blood samples. The competitive IAIP prototype rapid testdiscussed below utilized a diluted sample (1:5), which mitigated theneed for raw sample volume entering the test strip and containedblocking proteins and surfactants that mitigate non-specific bindingissues. A formulation-based sample pad (e.g., dried formula) can bedeveloped for components (e.g., protein blockers, surfactants, etc.) andtheir concentrations. There are available immunoassay additives that maybe used to effectively and reliably reduce interferences andnon-specific binding. Screening studies of candidate agents can befollowed by multi-factorial DoE to determine the optimal concentrationsof, e.g., blocking agents.

Conjugation techniques for LPS and heparin can be evaluated for use withgold colloid (nanoparticles) and dyed latex particle-based conjugationtechniques.

c. Blood Separation Filtration Membrane and Plastic Housing Cassette.

In addition to the dried sample pad formula, an in-line blood separationfilter membrane suitable for excluding red blood cells can be adoptedfor whole blood samples. The goal is to simplify the process and makethe testing of the rapid test format user friendly, especially for theoff-laboratory applications at point-of-care, such when using directlyapplied whole blood collected from finger prick or via collection tube.Multiple types of these blood filters are commercially available, suchas Vivid™ or Cytosep™ membranes (Pall) or other manufacturers (GELifesciences) and can be used in the LFIA. The plastic housingdimensions for the test strip can be selected for use with a reader(e.g., a Detekt reader).

We have determined that the hand-held Detekt™ reader model RDS-150 PRO(Detekt Biomedical, Austin, Tex.) can be used in the LFIA. Detekt'soptical lateral flow reader technology for rapid test readers iscurrently used in commercial diagnostic devices in food and beveragesafety testing, bio-threat detection, environmental monitoring andanimal health. The reader is a hand-held device that optically scans thetest strip and compares the Test and Control line signal strength to aprogrammed calibration algorithm. The signal integration software can becustomized to interpret the IAIP dose-response curve, and this algorithmcan be resident on Detekt™ units. The Vision Suite Pro developer's kitsoftware program can be used in collaboration with the manufacturer,Detekt Biomedical LLC in Austin, Tex., to integrate the interpretationof the IAIP test strip with the Detekt reader. A software algorithmbased on IAIP binding curve shape and internal controls can be developedto perform the following functions: a) to establish the shape of theIAIP binding curve relative to Test Line signal intensity; b) toestablish an internal control, possibly based on Control Line signal, tocorrect for day-to-day variability of test strip runs and c) toestablish the software for kit lot-specific information to beincorporated into the reader (e.g. via bar code scanning).

The performance of a developed and optimized sandwich-type IAIP rapidtest (according to the parameters discussed above and herein) can beevaluated in a pre-clinical setting. The LFIA cassette can be tested asfollows:

a) Analytical Sensitivity, Reportable Range and Precision

Samples/controls of known IAIP concentrations spanning the probablerange of IAIP concentrations (25-800 μg/mL) can be tested in multiplereplicates over multiple days (N=10) by multiple operators (N=3minimum). Data can be analyzed to establish precision at all spots ofthe IAIP binding curve, including precision of the assay cut-off point(set at 150 μg/mL).

b) Interfering Substances

Sample pools of known IAIP concentrations can be spiked with potentiallyinterfering substances to determine effects on dose recovery. TheClinical and Laboratory Standards Institute (CLSI) EP-07A protocol canbe used for reference. Stock solutions of substances (e.g. hemoglobin,bilirubin, human IgGs, etc.) that may be found at high concentrations inpatient samples can be spiked into IAIP samples and tested.

c) User Robustness Assessment—

Sample pools of known IAIP concentration can be used to verify therobustness of the test strip to various scenarios/errors produced byend-users. Parameters tested include: 1) incorrect sample volume added(+25%), 2) laboratory environmental conditions (temperature 65-85° F.;humidity 10-70% RH) and 3) incorrect test strip read time (+50-100%recommended).

d) Product Shelf-Life Stability—

IAIP test kits can be stored under different temperature regimens (e.g.,ambient and 37° C.) and tested using a set of IAIP controls of knownIAIP concentration at various intervals post-manufacture (>1 year) todetermine the stability of the product.

e) Sensitivity and Specificity in Preliminary Clinical Samples—

The product can be tested using clinical samples from in subjects (e.g.,infants with suspected and confirmed diagnoses of NS and NEC).

To evaluate the IAIP rapid test in an observational clinical study,samples can be collected from subjects (e.g., subjects having or at riskfor an inflammatory disease or condition or an infection) undergoingroutine clinical evaluation and management. Acquisition of data frommultiple centers caring for a broad, heterogeneous population ofsubjects with the same inflammatory disease or condition or infectioncan be used to provide an adequate number of study subjects andstrengthen the association between IAIP and the disease or infection.The rapid, point-of-care bedside device that can provide IAIP levels inreal time can aid in obviating the use of unnecessary interventions orthe prolongation of unnecessary treatment (e.g., antibiotic therapy) andcan reduce the length/cost of hospitalization.

The IAIP LFIA can be used for the clinical evaluation of subjects asfollows:

1) Collection of Blood Samples:

Samples can be collected from subjects having or at risk of developingan inflammatory disease or condition at a variety of medical centers.Clinical and demographic data can be recorded for all subjects,including age, weight, gender, and laboratory results. Serial samplesfrom the same subjects collected at various time points (e.g., 0, 24,48, 72 hrs. and 7d) can also be used in the LFIA. The LFIA could be usedto determine whether IAIP levels correlate with the severity andprogression of the disease, whether the IAIP level predicts risk orsubsequent disease, and whether subjects will develop more severedisease or improve following therapy.

2) Power Analysis:

Preliminary data and published studies of subjects having aninflammatory disease or condition or an infection can be used for samplesize estimates.

3) Blood Analysis:

The collected clinical plasma samples can be transferred into studyvials, labeled (deidentified), and stored in a frozen state untiltesting. IAIP levels are stable for 24 hours at room temperature, for upto 14 days under routine refrigerated clinical storage, and for anunlimited time at 20° C. IAIP levels can be analyzed using the LFIArapid IAIP test and the sandwich-type IAIP ELISA for a comparison study.

To evaluate proof of concept for an IAIP rapid test LFIA, we producedtest strip cassettes designed to measure IAIP using a competitive LFIAsimilar to that shown in FIG. 2A. The data produced using thisimmunoassay format, described below, confirm that a LFIA based on asandwich-type format (similar to that shown in FIG. 3A) can be used, aswell. The competitive LFIA is described below.

Sepsis and Systemic Inflammatory Response Syndrome in Neonates

Advances in intensive care have led to substantial improvement insurvival of subjects, (e.g., infants, especially preterm, very low birthweight (VLBW) (<1,500 g) infants). Preterm infants, however are prone toopportunistic infections and the acute life-threatening conditionsneonatal sepsis (NS) and necrotizing enterocolitis (NEC). A recentmulticenter survey suggests that up to 21% of VLBW infants encounter atleast one episode of late-onset (>72 hours of life) blood culture—provensepsis and up to 7% of VLBW develop NEC. NS and NEC are associated withserious morbidity, including adverse neuro-developmental outcomes, andhave a relatively high mortality in NS (10-30%) and NEC (16-42%). Earlywarning signs and symptoms of these neonatal diseases, are non-specific,frequently inconspicuous, and can easily be mistaken as due tonon-infectious etiologies, such as exacerbations of bronchopulmonarydysplasia, apnea of prematurity, gastroesophageal reflux, or functionalintestinal dysmotility. More disturbing is that the clinicaldeterioration in both diseases may progress in a fulminant mannerresulting in shock, disseminated intravascular coagulation, and deathwithin hours of clinical presentation. There are currently no rapidtests which broadly guide the physician as to the risk of patientprogression to severe disease and death.

Although the exact cause of NEC is still unclear, it is widely believedthat NEC pathogenesis results when infectious agents translocate acrossthe intestinal epithelial layer, evade innate immune defenses and causesubsequent inflammation and tissue necrosis. Both NS and NEC areassociated with systemic inflammatory responses. Their clinicalpresentation, which is non-specific and subtle at the initial stage, isvery similar. Furthermore, NS and NEC often coexist in the same diseaseepisode (for example, NEC with sepsis occurred in one-third of cases ofNEC in the case-control studies). Immediate medical management withprompt antimicrobial treatment and supportive care are standard of carefor both conditions. Thus, it is of practical and clinical importance toidentify the risk of these potentially lethal conditions at the earliestopportunity.

A biomarker for early and accurate identification of an inflammatorydisease or infection (e.g., NS and/or NEC) would be very useful to helpphysicians to make challenging decisions on initial use and continuationor early termination of antibiotic treatment in subjects (e.g., infants)in whom conventional tests are uninformative. Unfortunately, there iscurrently no widely-available biomarker that is clinically useful andeffective in the management of these challenging diseases. Recentlyprocalcitonin (PCT) has been approved as a biomarker to help manageantibiotic treatment in patients with lower respiratory tract infectionsand sepsis but this marker is specific only to bacterial infection andis not sensitive in detecting systemic inflammatory conditions caused byviral or other non-bacterial infections. Furthermore, the use of PCTtest is still controversial in pediatric patients especially in preterminfants during the first few days of life. Thus, a sensitive biomarkerthat provides information on the severity of the inflammatory diseaseprocess would be clinically useful in the management of inflammatorydiseases or infections (e.g., NS and NEC in preterm infants). Thisremains a major challenge.

Comparison Studies of the Predictive Value of IAIP with Other Markers inDetecting Infants with Systemic Inflammation and in a More ‘Localized’Disease (Spontaneous Intestinal Perforation)

We confirm that IAIP is an excellent severity biomarker and that it candifferentiate NEC from a more focal inflammatory disease, SpontaneousIntestinal Perforation (SIP). The IAIP test outperforms CRP in diagnosisof NEC from SIP.

IAIP can discern disease severity, e.g., a local inflammatory response(SIP) vs systemic and potentially life threatening inflammatoryresponses (NEC and NS). We collected blood samples from 95 infants (64female and 31 male) suspected of neonatal sepsis (NS), Necrotizingenterocolitis (NEC) and Spontaneous intestinal perforation (SIP) atWomen & Infants' Hospital in Providence, R.I. Serial samples were alsocollected when available from each individual patient. Most of theinfants were <30 weeks gestation age (ranging from 23-31 wks.) with meanbirth weight of 1235 grams. From these collected samples, we obtained 8infants with proven NEC, 9 infants with SIP and 20 infants with NS. Aswe have previously used the IAIP assay to determine the positive andnegative predictive value for NS, we assessed the predictive value ofIAIP levels in systemic inflammatory diseases conditions such as NEC andin a more ‘localized’ and focal necrotic disease condition, such as SIP.We also compared the levels of IAIP and another inflammatory biomarkerC-reactive protein (CRP) in these infants against gender, weight andgestational age matched controls. The competitive ELISA using a singlemonoclonal antibody specific against human IAIP (MAb 69.26) was used tomeasure the level of blood IAIP and the CRP ELISA kit was used toanalyze the CRP level in blinded fashion. A significant decrease in IAIPlevels was found in infants diagnosed with NEC (modified Bell's stage IIor higher) compared to healthy infants (p<0.05) and infants with SIP(with no radiographic NEC, p<0.005). However, no significant differencewas found between infants with SIP and the healthy controls. Incontrast, increased levels of CRP were found in both infants with NECand SIP, although the increase was not statistically significant(p>0.05) (see FIGS. 15A-15B). Furthermore, the receiver operatingcharacteristic (ROC) of CRP at a cutoff value of <4 generated the areaunder curve (AUC) of 0.65 (p=0.01, 95% Cl; 0.54-0.90) with sensitivityof 100%, specificity of 64.7%, positive predictive value (PPV) of 18.7and negative predictive value (NPV) of 100; while ROC of IAIP at acutoff value of <207 mg/L yielded robust AUC of 0.98 (p<0.0001, 95% Cl;0.84-0.99). The predictive value of IAIP was superior compared to thatof CRP with sensitivity of 100%, specificity of 88.2%, PPV of 41, andNPV of 100 (FIGS. 16A-16B).

Multiple serial samples from infants were available from some but notall infants before and after the onset of NEC and SIP. We furtheranalyzed the IAIP levels using the established IAIP competitive ELISA.The IAIP levels in infants with NEC were significantly lower compared tothe level found in SIP patients (FIG. 17) at diagnosis and following theinitiation of treatment. These were convenience samples that were onlyavailable from residual blood collections. These results demonstratethat IAIP level is a useful biomarker that identifies life threateningsystemic inflammatory conditions such as NEC (in addition to NS) withhigh sensitivity and specificity, and IAIP levels appear to alsodistinguish NEC from the less life-threatening conditions in SIPpatients. The IAIP test demonstrated excellent NPV both in NEC (100%)and NS (98%). The IAIP test can be used to guide treatment (e.g.,antibiotic treatment) decisions, such as early termination in infants inwhom conventional tests are uninformative.

Prototype Development of a Quantitative Competitive Lateral FlowImmunoassay (LFIA) for IAIP that Measures Blood IAIP Level and DetectsNS and NEC

a) Choice of Test Format and Assay Architecture:

A lateral flow immunoassay-based test was chosen for the format of IAIPrapid test (see FIG. 18). An LFIA offers many advantages when developinga rapid point-of-care assay as they are designed to: 1) use small samplevolumes; 2) follow well characterized kinetics and offer rapid intervalsto test results; 3) be analytically sensitive and precise; 4) commonlycontain an internal control to verify proper performance of the device;5) be made from raw materials that are well characterized and widelyavailable; and 6) produce signals that can be quantitatively analyzedusing a strip reader. Thus, LFIA, as an IAIP rapid test, can be used,e.g., at a point-of-care setting.

b) Optimization of Capture, Detection and Sample Reagents and Conditionsof the LFIA:

Initial efforts were successful in adopting the established competitiveIAIP ELISA to the competitive LFIA format by optimizing the capture,detection, sample reagents and conditions of the lateral flowimmunoassay. We further established a dose-response curve, as well asevaluated the timing of signal, precision, and repeatability of therapid IAIP assay format. A reader-based data analysis of test resultswere initially adopted using a Qiagen test strip reader. Following theseinitial tests, we have proceeded to optimize the test strips and assayconditions, and test several portable readers that available andsuitable for our test strips.

c) Purified IAIP and Monoclonal Antibodies Against IAIP (MAb 69.26):

Both reagents are key components used in the competitive assay format.We have developed and optimized a scalable bioprocess method forisolation of IAIP from human plasma that results in high yield and highpurity. The hybridoma cells were grown in scalable CELLine cultureflasks (Integra Bioscience) for large scale production of antibodies invitro. The IgG was isolated from the hybridoma supernatant by affinityProtein A chromatography.

d) Test Strip:

Multiple pilot lots of strips were prepared and the chosen formula is astable platform for the quantitative measure of IAIP in human plasma: 1)Sample pad: cellulose pad with buffer and surfactant; 2) Conjugate pad:glass fiber pad with anti-IAIP (MAb 69.26) gold conjugate and rabbit IgGgold conjugate (Control Line); 3) Nitrocellulose: Fast flow (MilliporeHF090) nitrocellulose with IAIP striped (Test Line) and goat-anti-RabbitIgG striped (Control Line Terminal wick: cellulose pad (no formula)).

e) Sample:

A small volume of plasma (15 μl of 1:5 diluted samples) was added tostrip then 115 μl chase buffer to push sample through strip. The chasebuffer had been optimized: 2 mM Tris pH 8.0+100 mM NaCl+0.5% Brij+0.05%Tween 20+1.0% Fetal Calf Serum. Brij surfactant was added to the bufferto facilitate a rapid clearing of the conjugate from the upstreamportions of the strip

f) Running Time:

Strips were run with plasma samples or IAIP calibrators (at threedifferent concentrations) and then quantified by the Qiagen reader at10, 15, 20, 25, 30, 45 and 60 minutes after sample addition. The stripran in about 7 min (i.e., nitrocellulose had cleared of extraneous gold)and could be read any time after that. We found that after 15 min thesignals did not change dramatically. Thus, we read the strips uniformlyat 15 min after the samples were added.

g) Calibrators and Controls:

Human plasma (Rhode Island Blood Center) with a known IAIP value basedon the ELISA results was used as calibrator and internal standardcontrol. Different doses were created by diluting the plasma with 1%FCS-containing buffer. Using a portable Qiagen test reader (ESEQuantLFR) we established a standard curve that ranged from 17.5 to 1100 μg/mL(using 1:5 sample dilution). Reproducibility of calibration curvesacross days was tested by repeating and running a set of the calibratorsup to 13 times at different times of the day over 5 days. The resultsare shown in FIG. 19. The mean and the SD of each of the values wereplotted. The results show that the test demonstrated a tight spread overthe 5 day period with an excellent coefficient of variance (CV less than15%).

h) Test Strip Readers and Software:

As our IAIP rapid test is intended to be quantitative, we carefullyselected a reader to capture the signal generated by the test strips.Various types of test strip readers are commercially available. Thechoice of reader was based on features and specifications such asengineering, ergonomics, and software robustness. Since this is animportant part of the rapid test, we tested three different readers withdifferent technologies and features for IAIP rapid test strips: 1) aportable tabletop lateral flow reader (ESEQuant LFR by Qiagen); 2) ahandheld PDA based reader Detekt RDS 1500 Pro (Detekt Biomedical,Austin, Tex.) and 3) Smart Phone/tablet based reader system (iCalqreader—iCalq, Salt Lake City, Utah). We found that the Detekt readerproduced a better linear curve ranging from 5 to 700 μg/mL than theother readers tested.

Determination of Inter- and Intra-Assay Precision of the Optimized Assay

The inter-assay precision characteristics of the rapid test weredetermined using collected blood samples (n=6) run repeatedly for 6consecutive days. The results were read subsequently using threedifferent portable and handheld readers and the coefficient of variance(CV) was calculated from the results obtained from each individualsamples. The CV of the IAIP rapid test using ESEQuant reader rangedbetween 4 to 16% with an average of 13%, while the Detekt readerdemonstrated an average CV of 10% (ranging from 5-16%) and the iCalQsmart phone based reader resulted in an average CV of 16% (ranging from10-23%). The Detekt reader performed better with a relatively lower CVthan the other two readers we tested. In order to evaluate theintra-assay precision of the test, we ran 8 plasma samples and analyzedIAIP levels based on the optimized protocols. The signal obtained fromeach sample was read three times on the various readers and IAIP valueswere calculated individually based on the established standard curves.Subsequently, the CV was determined from the triplicate readings and wasfound to range between 2 to 8% with an average CV of 5% for all threereaders tested.

Performance Assessment of the Rapid Assay in Infant Plasma

Using the samples collected from infants at Women & Infants' Hospital,we carried out the analysis of IAIP based on the optimized conditionsfor the IAIP test strips described above. The resultant signals wereread sequentially using three independent readers and the results werecalculated based on the respective standard curve of the readers. Theresults of the rapid tests were compared with the results obtained bythe established competitive ELISA assay and the values of eachindividual sample were plotted against the results of the rapid test(FIGS. 20A-20C). The results demonstrated an excellent correlationbetween the 6 hr competitive ELISA and the rapid competitive LFIA testresults that were generated within 15 minutes by using the ESEQuantreader (Correlation coeff. R2 of 0.832, n=311) and Detekt reader(R2=0.84, n=339). However, the iCalQ smart phone based reader producedtest results that were less comparable with the ELISA results,especially in the samples containing a high level IAIP (>600 μg/ml).

In summary, we successfully converted a 6 hour laboratory-basedcompetitive ELISA into a point-of-care IAIP rapid test that is capableof measuring circulating IAIP in blood (ranging from 10-800 μg/mL)accurately (correlation coefficient R2>0.8 compare to the ELISA results)within 15 minutes with acceptable intra- and inter-assay precision (lessthan 20% CV). We confirmed that blood IAIP level is a useful predictivemarker not only for NS, but also for NEC with high sensitivity (100%)and high specificity (88%). The IAIP test is more specific in detectingNEC than the CRP test and IAIP level is also useful in distinguishingNEC from SIP patients.

Given the successful adaptation of the competitive assay to the LFIAformat, we expect that the “sandwich-type” IAIP ELISA described herein(e.g., as shown in FIG. 3A) can also be adapted to LFIA format. Rapidquantification of IAIP can lead to early identification of subjects(e.g., human subjects, such as infants, children, adolescents, oradults) having or at risk of developing an inflammatory disease orcondition or an infection (e.g., sepsis, NEC, bacterial infection, oranother disease or condition) and timely initiation of optimal therapy.

Example 11: Hyaluronic Acid-IAIP Solid Binding Assay

Hyaluronic Acid, sodium salt (Sigma-Aldrich), was dissolved in dH₂O(stock concentration of 1 mg/mL) and further diluted into 20 mMNaHCO₃/Na₂CO₃ buffer at pH 9.0. Fifty, 100 and 200 ng hyaluronic acidper well was immobilized on a 96-well microplate (Greiner BioOne,Microlon 600) at 37° C. for 120 minutes. Following blocking with 5%Non-fat Milk Powder in TBS-T (20 mM Tris-buffered saline solution at pH7.3+0.05% Tween-20 (v/v)) at 37° C. for 60 minutes and 3 washes withTBS-T, serially diluted solution containing IAIP (human plasma andhighly purified IAIP in TBS) was added to the microplate and incubatedat 37° C. for 60 minutes. Human plasma was prepared bycryo-precipitation of fresh frozen plasma obtained from a local bloodbank. The cryo-supernatant was used in this experiment and had an IAIPconcentration of 250 μg/mL. The purified IAIP was at a startingconcentration of 1 mg/mL. The microplate was then washed 3 times withTBS-T, and biotin-conjugated MAb 69.26 (monoclonal antibody againsthuman IAIP) at 1:1000 dilution in TBS was added and incubated at 37° C.for 30 minutes.

Following three additional washes with TBS-T, horseradish peroxidase(HRP)-conjugated Streptavidin (Inova Bioscience) diluted to 1:5000 inTBS was added and incubated at 37° C. for 30 minutes. After washing 3times with TBS-T, 50 μL Enhanced K-Blue TMB Substrate (Neogen) was addedto each well and the reaction was stopped by adding 50 μL 1Mhydrochloric acid (HCl). The color change was read using aspectrophotometer (Molecular Devices SpectraMax Plus microplate reader)at 650 nm wavelength, and the standard curve for human plasma orpurified IAIP was plotted at 50, 100 and 200 ng/well immobilizedhyaluronic acid (FIGS. 21A-21B). These data show that IAIP can bequantitatively measured in biological mixtures by capturing IAIP complexmolecules using hyaluronic acid and detecting the captured IAIP using amonoclonal antibody specific for the light chain of IAIP (e.g., MAb69.26). As shown in FIGS. 21A-21B, a standard curve can be optimized andestablished using purified IAIP or human plasma with a known amount ofIAIP. Consequently, this assay can be used to quantify an unknown amountof IAIP in a biological sample from a subject. An alternative approachto this method can be employed, in which an IAIP-specific monoclonalantibody is used to capture IAIP and biotin-labeled hyaluronic acid isused as a ligand to detect the bound IAIP, similar to the approachesused in Examples 1 and 2.

OTHER EMBODIMENTS

All publications, patents, and patent applications mentioned in theabove specification are hereby incorporated by reference to the sameextent as if each individual publication, patent or patent applicationwas specifically and individually indicated to be incorporated byreference in its entirety. U.S. Provisional Application Nos. 62/490,003and 62/614,333 are specifically incorporated herein in their entirety.Various modifications and variations of the described methods,pharmaceutical compositions, and kits of the invention will be apparentto those skilled in the art without departing from the scope and spiritof the claimed invention. Although the disclosure has been described inconnection with specific embodiments, it will be understood that it iscapable of further modifications and that the invention as claimedshould not be unduly limited to such specific embodiments.

1. A method for quantifying inter-alpha inhibitor protein (IAIP) in asample from a subject comprising: a) contacting the sample with abinding agent to produce an IAIP-binding agent complex, wherein thebinding agent is bound to a support; b) contacting the IAIP-bindingagent complex with a detection agent; and c) detecting an amount of thedetection agent bound to the IAIP-binding agent complex to quantify IAIPin the sample.
 2. The method of claim 1, wherein the IAIP is intactIAIP.
 3. The method of claim 1 or 2, wherein the binding agent is anIAIP ligand that binds to IAIP.
 4. The method of claim 1 or 2, whereinthe binding agent is an antibody that specifically binds to IAIP.
 5. Themethod of any one of claims 1 to 4, wherein the detection agentcomprises an IAIP ligand.
 6. The method of claim 5, wherein thedetection agent further comprises an antibody that binds to the IAIPligand detection agent.
 7. The method of any one of claims 1 to 4,wherein the detection agent is an antibody that specifically binds toIAIP.
 8. The method of claim 1, wherein the IAIP is in an IAIP-IAIPligand complex.
 9. The method of claim 8, wherein the binding agent isan IAIP ligand that binds to IAIP.
 10. The method of claim 9, whereinthe IAIP ligand of the IAIP-IAIP ligand complex is different from thebinding agent.
 11. The method of claim 8, wherein the binding agent isan antibody that binds to the IAIP ligand of the IAIP-IAIP ligandcomplex.
 12. The method of claim 8, wherein the binding agent is anantibody that specifically binds to IAIP of the IAIP-IAIP ligandcomplex.
 13. The method of any one of claims 8 to 12, wherein thedetection agent comprises an IAIP ligand that binds to IAIP.
 14. Themethod of claim 13, wherein the detection agent further comprises anantibody that binds to the IAIP ligand detection agent.
 15. The methodof claim 13 or 14, wherein the IAIP ligand of the IAIP-IAIP ligandcomplex is different from the IAIP ligand detection agent.
 16. Themethod of any one of claims 8 to 12, wherein the detection agent is anantibody that binds to the IAIP ligand of the IAIP-IAIP ligand complex.17. The method of any one of claims 8 to 12, wherein the detection agentis an antibody that specifically binds to IAIP of the IAIP-IAIP ligandcomplex.
 18. The method of any one of claims 4, 6, 7, 11, 12, 14, 16,and 17, wherein the antibody is a monoclonal antibody.
 19. The method ofany one of claims 4, 7, 12, and 17, wherein the antibody is MAb 69.26 orMAb 69.31.
 20. The method of any one of claims 3, 5, 6, and 8 to 17,wherein the IAIP ligand is selected from the group consisting ofendotoxin (LPS), heparin, a histone, hyaluronic acid, vitronectin,fibronectin, laminin, tenascin C, aggrecan, von Willebrand Factor,pentraxin-3 (PTX3), TNF-stimulated gene-6 (TSG-6), factor IX, acomplement component, factor XIIIa, and tissue transglutaminase.
 21. Themethod of claim 28, wherein the complement component is C1q, C2, C3, C4,C5, C6, C8, properdin, or factor D.
 22. The method of any one of claims1 to 21, wherein the detection agent comprises a label.
 23. The methodof claim 22, wherein the label is biotin, an enzyme, an enzymesubstrate, a radiolabel, a luminescent compound, colloidal gold, aparticle, or a fluorescent dye.
 24. The method of any one of claims 1 to23, wherein the support is a plate, a particle, a nanoparticle, a resin,a membrane, a biochip, a container, a test strip, or a bead.
 25. Themethod of any one of claims 1 to 24, wherein the method further includesa wash step between steps a) and b).
 26. The method of any one of claims1 to 25, wherein the method further includes a wash step between stepsb) and c).
 27. The method of any one of claims 1 to 26, wherein themethod further includes a blocking step prior to step a) or step b). 28.The method of any one of claims 1 to 27, wherein the contacting in stepa) and/or b) is performed at a pH of about 7.0 to about 3.5.
 29. Themethod of claim 28, wherein the pH is about 5.0 to about 3.5.
 30. Themethod of claim 29, wherein the pH is about 4.0.
 31. The method of anyone of claims 1 to 30, wherein the sample is a fluid.
 32. The method ofclaim 31, wherein the fluid is blood, plasma, serum, urine,cerebrospinal fluid, synovial fluid, amniotic fluid, interstitial fluid,follicular fluid, peritoneal fluid, bronchoalveolar lavage fluid, breastmilk, sputum, lymph, bile, or tissue homogenate.
 33. The method of anyone of claims 1 to 32, wherein the subject is a human subject.
 34. Themethod of claim 33, wherein the subject has been identified as having orat risk of developing an inflammatory disease or condition or aninfection.
 35. The method of claim 33, wherein the subject has not beenidentified as having or at risk of developing an inflammatory disease orcondition or an infection.
 36. The method of claim 33, wherein themethod is performed before, after, or concurrent with diagnosis of thesubject as having or at risk of an inflammatory disease or condition oran infection.
 37. The method of claim 33 or 34, wherein the method isperformed substantially concurrent with treatment of the subject for aninflammatory disease or condition or an infection.
 38. The method of anyone of claims 33 to 36, wherein the method is performed prior totreatment of the subject for an inflammatory disease or condition or aninfection.
 39. The method of claim 33 or 34, wherein the method isperformed after treatment of the subject for an inflammatory disease orcondition or an infection.
 40. The method of any one of claims 34 to 39,wherein the inflammatory disease or condition is selected from the groupconsisting of sepsis, septic shock, sterile sepsis, trauma, injury,stroke, acute inflammatory disease, SIRS, acute lung injury, ARDS,pneumonia, necrotizing enterocolitis, acute pancreatitis, renal disease,acute kidney injury, liver injury, acute circulatory failure,preeclampsia, cancer, cancer metastasis, tumor invasion, peripheralartery disease, type 1 or type 2 diabetes, atheroscleroticcardiovascular disease, intermittent claudication, critical limbischemic disease, myocardial infarction, carotid occlusion, umbilicalcord occlusion, low birth-weight, premature birth, surgery-inducedinflammation, abscess-induced inflammation, multiple sclerosis,pulmonary insufficiency, peripheral neuropathy, hypoxic ischemia,bacterial infection, wounds, burns, lacerations, contusions, bonefractures, surgical procedures, tissue ischemia, rheumatoid arthritis,meningitis, inflammatory bowel disease, chronic obstructive pulmonarydisease, rhinitis, preterm labor, or an infectious disease.
 41. Themethod of any one of claims 34 to 39, wherein the infection is caused bya gram negative bacteria, such as Neisseria species including Neisseriagonorrhoeae and Neisseria meningitidis, Branhamella species includingBranhamella catarrhalis, Escherichia species including Escherichia coli,Enterobacter species, Proteus species including Proteus mirabilis,Pseudomonas species including Pseudomonas aeruginosa, Pseudomonasmallei, and Pseudomonas pseudomallei, Klebsiella species includingKlebsiella pneumoniae, Salmonella species, Shigella species, Serratiaspecies, Acinetobacter species; Haemophilus species includingHaemophilus influenzae and Haemophilus ducreyi, Brucella species,Yersinia species including Yersinia pestis and Yersinia enterocolitica,Francisella species including Francisella tularensis, Pasteurellaspecies including Pasteurella multocida, Vibrio cholerae, Flavobacteriumspecies, meningosepticum, Campylobacter species including Campylobacterjejuni, Bacteroides species (oral, pharyngeal) including Bacteroidesfragilis, Fusobacterium species including Fusobacterium nucleatum,Calymmatobacterium granulomatis, Streptobacillus species includingStreptobacillus moniliformis, and Legionella species includingLegionella pneumophila.
 42. The method of any one of claims 1 to 41,wherein the subject is a neonate, a child, an adolescent, or an adult.43. The method of any one of claims 1 to 42, wherein the method isperformed one or more times per year.
 44. The method of claim 43,wherein the method is performed one or more times per month.
 45. Themethod of claim 44, wherein the method is performed one or more timesper week.
 46. The method of claim 45, wherein the method is performedone or more times per day.
 47. The method of claim 46, wherein themethod is performed one or more times per hour.
 48. The method of anyone of claims 1 to 47, wherein the method is performed at least once, atleast twice, at least three times, at least five times, or at least tentimes.
 49. The method of any one of claims 1 to 48, wherein the methodfurther comprises administering a treatment comprising IAIP or atherapeutic agent to the subject.
 50. The method of claim 49, whereinthe subject has an IAIP concentration of 200 μg/mL or lower.
 51. Themethod of claim 49, wherein the sample from the subject has an elevatedlevel of IAIP-IAIP ligand complex relative to a reference sample. 52.The method of any one of claims 49 to 51, wherein the subject has or isat risk of developing an inflammatory disease or condition or aninfection.
 53. The method of any one of claims 49 to 52, wherein themethod comprises administering IAIP and a therapeutic agent to thesubject.
 54. The method of any one of claims 49-53, wherein thetherapeutic agent is selected from the group consisting of an antibioticagent, an antiviral agent, an antifungal agent, an antiparasitic agent,an anti-inflammatory agent, an anti-cancer agent, an anti-coagulant, animmunomodulatory agent, a bronchodilator agent, a complement inhibitor,a vasopressor, a sedative, or mechanical ventilation.
 55. The method ofany one of claims 1 to 54, wherein the subject has been ill for at leastone day.
 56. The method of claim 55, wherein the subject has been illfor at least one week.
 57. The method of claim 56, wherein the subjecthas been ill for at least one month.
 58. The method of claim 57, whereinthe subject has been ill for at least one year.
 59. The method of anyone of claims 1 to 58, wherein the method is for: a) evaluating thehealth status of the subject; b) monitoring the health status of thesubject; c) diagnosing the subject as having or being at risk for aninflammatory disease or condition or an infection; d) evaluatingefficacy of a treatment administered to the subject; or e) evaluatingdisease severity in the subject.
 60. The method of claim 59, wherein themethod further comprises comparing the amount of IAIP and/or anIAIP-IAIP ligand complex detected in the sample to the amount of IAIPand/or an IAIP-IAIP ligand complex found in a sample from a normalsubject or to a cutoff value.
 61. The method of claim 60, wherein anamount of IAIP in the sample that is lower than an amount of IAIP in thesample from the normal subject or relative to the cutoff value indicatesthat the subject has or is at risk of developing an inflammatory diseaseor condition or an infection.
 62. The method of claim 60, wherein anamount of an IAIP-IAIP ligand complex in the sample that is greater thanan amount of IAIP-IAIP ligand complex in the sample from the normalsubject or relative to the cutoff value indicates that the subject hasor is at risk of developing an inflammatory disease or condition or aninfection.
 63. The method of any one of claims 60 to 62, wherein theamount of IAIP in the sample from the normal subject, or the cutoffvalue, is >250 μg/mL.
 64. The method of claim 63, wherein the amount ofIAIP in the sample from the normal subject is about 260 to about 540μg/mL.
 65. The method of any one of claims 1 to 64, wherein adetermination that the subject has an IAIP concentration of 250 μg/mL orless indicates that the subject has or is at high risk of developing aninflammatory disease or condition or an infection or is diagnosed ashaving an increased risk of morbidity and/or mortality.
 66. The methodof any one of claims 60 to 65, wherein the subject has an IAIPconcentration of 200 to 300 μg/mL and wherein the method is performed atleast once a year.
 67. The method of claim 66, wherein the method isperformed at least twice a year.
 68. The method of claim 66, wherein themethod is performed at least once a month.
 69. The method of claim 66,wherein the method is performed at least once a week.
 70. The method ofclaim 66, wherein the method is performed at least once a day.
 71. Themethod of claim 66, wherein the method is performed at least once anhour.
 72. The method of any one of claims 1 to 71, wherein the subjectpreviously had an inflammatory disease or condition or an infection. 73.The method of any one of claims 49 to 54, wherein the method isperformed prior to the treatment and one or more times during the courseof the treatment.
 74. The method of any one of claims 49 to 73, whereinthe method is performed after initiation of the treatment and/or afterconclusion of the treatment.
 75. The method of any one of claims 49 to74, wherein the treatment is determined to be effective if theconcentration of IAIP increases in the subject relative to a priormeasurement of IAIP in the subject and/or if the concentration of anIAIP-IAIP ligand complex decreases in the subject relative to a priormeasurement of an IAIP-IAIP ligand complex in the subject.
 76. Themethod of any one of claims 49 to 74, wherein the treatment isdetermined to be ineffective if the concentration of IAIP decreases orremains constant in the subject relative to a prior measurement of IAIPin the subject and/or if the concentration of an IAIP-IAIP ligandcomplex increases or remains constant in the subject relative to a priormeasurement of an IAIP-IAIP ligand complex in the subject.
 77. Themethod of claim 76, wherein the method further comprises modifying orchanging the treatment.
 78. A method of treating a subject that has oris at risk of developing an inflammatory disease or infection, whereinthe subject has been determined to be in need of treatment according tothe method of any one of claims 1-77, comprising administering to thesubject a therapeutically effective amount of IAIP and/or a therapeuticagent selected from the group consisting of an antibiotic agent, anantiviral agent, an antifungal agent, an antiparasitic agent, ananti-inflammatory agent, an anti-cancer agent, an anti-coagulant, animmunomodulatory agent, a bronchodilator agent, a complement inhibitor,a vasopressor, a sedative, or mechanical ventilation.
 79. The method ofclaim 78, wherein the inflammatory disease or condition is selected fromthe group consisting of sepsis, septic shock, sterile sepsis, trauma,injury, stroke, acute inflammatory disease, SIRS, acute lung injury,ARDS, pneumonia, necrotizing enterocolitis, acute pancreatitis, renaldisease, acute kidney injury, liver injury, acute circulatory failure,surgery-induced inflammation, abscess-induced inflammation, multiplesclerosis, preeclampsia, cancer, cancer metastasis, tumor invasion,peripheral artery disease, type 1 or type 2 diabetes, atheroscleroticcardiovascular disease, intermittent claudication, critical limbischemic disease, myocardial infarction, carotid occlusion, umbilicalcord occlusion, low birth-weight, premature birth, pulmonaryinsufficiency, peripheral neuropathy, hypoxic ischemia, bacterialinfection, wounds, burns, lacerations, contusions, bone fractures,surgical procedures, tissue ischemia, rheumatoid arthritis, meningitis,inflammatory bowel disease, chronic obstructive pulmonary disease,rhinitis, preterm labor, or an infectious disease.
 80. The method ofclaim 78, wherein the infection is caused by a gram negative bacteria,such as Neisseria species including Neisseria gonorrhoeae and Neisseriameningitidis, Branhamella species including Branhamella catarrhalis,Escherichia species including Escherichia coli, Enterobacter species,Proteus species including Proteus mirabilis, Pseudomonas speciesincluding Pseudomonas aeruginosa, Pseudomonas mallei, and Pseudomonaspseudomallei, Klebsiella species including Klebsiella pneumoniae,Salmonella species, Shigella species, Serratia species, Acinetobacterspecies; Haemophilus species including Haemophilus influenzae andHaemophilus ducreyi, Brucella species, Yersinia species includingYersinia pestis and Yersinia enterocolitica, Francisella speciesincluding Francisella tularensis, Pasteurella species includingPasteurella multocida, Vibrio cholerae, Flavobacterium species,meningosepticum, Campylobacter species including Campylobacter jejuni,Bacteroides species (oral, pharyngeal) including Bacteroides fragilis,Fusobacterium species including Fusobacterium nucleatum,Calymmatobacterium granulomatis, Streptobacillus species includingStreptobacillus moniliformis, and Legionella species includingLegionella pneumophila.
 81. A kit for quantifying IAIP or an IAIP-IAIPligand complex in a sample, wherein the kit comprises an IAIP bindingagent and an IAIP detection agent and, optionally, one or more of thefollowing: a wash buffer, a blocking agent, a substrate for detection ofa label, and instructions for quantifying a level of IAIP or anIAIP-IAIP ligand complex in a sample.
 82. The kit of claim 81, whereinthe binding agent is immobilized on a support.
 83. The kit of claim 81or 82, wherein the detection agent is labeled.
 84. The kit of any one ofclaims 81 to 83, wherein the IAIP binding agent is an IAIP-specificantibody or an IAIP ligand.
 85. The kit of any one of claims 81 to 84,wherein the kit further comprises an IAIP ligand binding agent.
 86. Thekit of claim 85, wherein the IAIP ligand binding agent is an antibodythat binds to an IAIP ligand.
 87. The kit of any one of claims 81 to 86,wherein the IAIP detection agent is an IAIP-specific antibody or an IAIPligand.
 88. The kit of any one of claims 81 to 87, wherein the kitfurther comprises an IAIP ligand detection agent.
 89. The kit of claim88, wherein the IAIP ligand detection agent is an antibody that bindsspecifically to an IAIP ligand.
 90. The kit of claim 84 or 87, whereinthe IAIP-specific antibody is a monoclonal antibody.
 91. The kit ofclaim 90, wherein the monoclonal antibody is MAb 69.26 or MAb 69.31. 92.The kit of any one of claims 82 to 91, wherein the support is a plate, aresin, a container, a membrane, a biochip, a particle, a nanoparticle, atest strip, or a bead.
 93. The kit of any one of claims 81 to 92,wherein the label is an enzyme, an enzyme substrate, biotin, a particle,a fluorescent dye, a luminescent compound, or a radiolabel.
 94. The kitof any one of claims 84, 86, 87, 88, and 89, wherein the IAIP ligand isselected from the group consisting of endotoxin (LPS), heparin, ahistone, hyaluronic acid, laminin, tenascin C, aggrecan, vitronectin,fibronectin, von Willebrand Factor, pentraxin-3 (PTX3), TNF-stimulatedgene-6 (TSG-6), factor IX, a complement component, factor XIIIa, andtissue transglutaminase.
 95. The method of claim 1, wherein the bindingagent is an IAIP ligand that binds to IAIP.
 96. The method of claim 1,wherein the binding agent is an antibody that specifically binds toIAIP.
 97. The method of claim 1, 95 or 96, wherein the detection agentcomprises an IAIP ligand.
 98. The method of claim 97, wherein thedetection agent further comprises an antibody that binds to the IAIPligand detection agent.
 99. The method of claim 1, 95, or 96, whereinthe detection agent is an antibody that specifically binds to IAIP. 100.The method of claim 8 or 11, wherein the detection agent comprises anIAIP ligand that binds to IAIP.
 101. The method of claim 100, whereinthe detection agent further comprises an antibody that binds to the IAIPligand detection agent.
 102. The method of claim 100, wherein the IAIPligand of the IAIP-IAIP ligand complex is different from the IAIP liganddetection agent.
 103. The method of claim 8, 9, or 12, wherein thedetection agent is an antibody that binds to the IAIP ligand of theIAIP-IAIP ligand complex.
 104. The method of claim 8 or 11, wherein thedetection agent is an antibody that specifically binds to IAIP of theIAIP-IAIP ligand complex.
 105. The method of claim 96, wherein theantibody is a monoclonal antibody.
 106. The method of claim 97, whereinthe antibody is a monoclonal antibody.
 107. The method of claim 99,wherein the antibody is a monoclonal antibody.
 108. The method of claim101, wherein the antibody is a monoclonal antibody.
 109. The method ofclaim 103, wherein the antibody is a monoclonal antibody.
 110. Themethod of claim 104, wherein the antibody is a monoclonal antibody. 111.The method of claim 96, wherein the antibody is MAb 69.26 or MAb 69.31.112. The method of claim 99, wherein the antibody is MAb 69.26 or MAb69.31
 113. The method of claim 104, wherein the antibody is MAb 69.26 orMAb 69.31
 114. The method of claim 95, wherein the IAIP ligand isselected from the group consisting of endotoxin (LPS), heparin, ahistone, hyaluronic acid, vitronectin, fibronectin, laminin, tenascin C,aggrecan, von Willebrand Factor, pentraxin-3 (PTX3), TNF-stimulatedgene-6 (TSG-6), factor IX, a complement component, factor XIIIa, andtissue transglutaminase.
 115. The method of claim 97, wherein the IAIPligand is selected from the group consisting of endotoxin (LPS),heparin, a histone, hyaluronic acid, vitronectin, fibronectin, laminin,tenascin C, aggrecan, von Willebrand Factor, pentraxin-3 (PTX3),TNF-stimulated gene-6 (TSG-6), factor IX, a complement component, factorXIIIa, and tissue transglutaminase.
 116. The method of claim 100,wherein the IAIP ligand is selected from the group consisting ofendotoxin (LPS), heparin, a histone, hyaluronic acid, vitronectin,fibronectin, laminin, tenascin C, aggrecan, von Willebrand Factor,pentraxin-3 (PTX3), TNF-stimulated gene-6 (TSG-6), factor IX, acomplement component, factor XIIIa, and tissue transglutaminase. 117.The method of claim 8, wherein the IAIP ligand is selected from thegroup consisting of endotoxin (LPS), heparin, a histone, hyaluronicacid, vitronectin, fibronectin, laminin, tenascin C, aggrecan, vonWillebrand Factor, pentraxin-3 (PTX3), TNF-stimulated gene-6 (TSG-6),factor IX, a complement component, factor XIIIa, and tissuetransglutaminase.
 118. The method of any one of claims 114-117, whereinthe complement component is C1q, C2, C3, C4, C5, C6, C8, properdin, orfactor D.
 119. The method of claim 1, wherein the detection agentcomprises a label.
 120. The method of claim 119, wherein the label isbiotin, an enzyme, an enzyme substrate, a radiolabel, a luminescentcompound, colloidal gold, a particle, or a fluorescent dye.
 121. Themethod of claim 1, wherein the support is a plate, a particle, ananoparticle, a resin, a membrane, a biochip, a container, a test strip,or a bead.
 122. The method of claim 1, wherein the method furtherincludes a wash step between steps a) and b).
 123. The method of claim1, wherein the method further includes a wash step between steps b) andc).
 124. The method of claim 1, wherein the method further includes ablocking step prior to step a) or step b).
 125. The method of claim 1,wherein the contacting in step a) and/or b) is performed at a pH ofabout 7.0 to about 3.5.
 126. The method of claim 125, wherein the pH isabout 5.0 to about 3.5.
 127. The method of claim 126, wherein the pH isabout 4.0.
 128. The method of claim 1, wherein the sample is a fluid.129. The method of claim 128, wherein the fluid is blood, plasma, serum,urine, cerebrospinal fluid, synovial fluid, amniotic fluid, interstitialfluid, follicular fluid, peritoneal fluid, bronchoalveolar lavage fluid,breast milk, sputum, lymph, bile, or tissue homogenate.
 130. The methodof claim 1, wherein the subject is a human subject.
 131. The method ofclaim 130, wherein the subject has been identified as having or at riskof developing an inflammatory disease or condition or an infection. 132.The method of claim 130, wherein the subject has not been identified ashaving or at risk of developing an inflammatory disease or condition oran infection.
 133. The method of claim 130, wherein the method isperformed before, after, or concurrent with diagnosis of the subject ashaving or at risk of an inflammatory disease or condition or aninfection.
 134. The method of claim 130, wherein the method is performedsubstantially concurrent with treatment of the subject for aninflammatory disease or condition or an infection.
 135. The method ofclaim 130, wherein the method is performed prior to treatment of thesubject for an inflammatory disease or condition or an infection. 136.The method of claim 130, wherein the method is performed after treatmentof the subject for an inflammatory disease or condition or an infection.137. The method of claim 131, wherein the inflammatory disease orcondition is selected from the group consisting of sepsis, septic shock,sterile sepsis, trauma, injury, stroke, acute inflammatory disease,SIRS, acute lung injury, ARDS, pneumonia, necrotizing enterocolitis,acute pancreatitis, renal disease, acute kidney injury, liver injury,acute circulatory failure, preeclampsia, cancer, cancer metastasis,tumor invasion, peripheral artery disease, type 1 or type 2 diabetes,atherosclerotic cardiovascular disease, intermittent claudication,critical limb ischemic disease, myocardial infarction, carotidocclusion, umbilical cord occlusion, low birth-weight, premature birth,surgery-induced inflammation, abscess-induced inflammation, multiplesclerosis, pulmonary insufficiency, peripheral neuropathy, hypoxicischemia, bacterial infection, wounds, burns, lacerations, contusions,bone fractures, surgical procedures, tissue ischemia, rheumatoidarthritis, meningitis, inflammatory bowel disease, chronic obstructivepulmonary disease, rhinitis, preterm labor, or an infectious disease.138. The method of claim 131, wherein the infection is caused by a gramnegative bacteria, such as Neisseria species including Neisseriagonorrhoeae and Neisseria meningitidis, Branhamella species includingBranhamella catarrhalis, Escherichia species including Escherichia coli,Enterobacter species, Proteus species including Proteus mirabilis,Pseudomonas species including Pseudomonas aeruginosa, Pseudomonasmallei, and Pseudomonas pseudomallei, Klebsiella species includingKlebsiella pneumoniae, Salmonella species, Shigella species, Serratiaspecies, Acinetobacter species; Haemophilus species includingHaemophilus influenzae and Haemophilus ducreyi, Brucella species,Yersinia species including Yersinia pestis and Yersinia enterocolitica,Francisella species including Francisella tularensis, Pasturella speciesincluding Pasteurella multocida, Vibrio cholerae, Flavobacteriumspecies, meningosepticum, Campylobacter species including Campylobacterjejuni, Bacteroides species (oral, pharyngeal) including Bacteroidesfragilis, Fusobacterium species including Fusobacterium nucleatum,Calymmatobacterium granulomatis, Streptobacillus species includingStreptobacillus moniliformis, and Legionella species includingLegionella pneumophila.
 139. The method of claim 1, wherein the subjectis a neonate, a child, an adolescent, or an adult.
 140. The method ofclaim 1, wherein the method is performed one or more times per year.141. The method of claim 140, wherein the method is performed one ormore times per month.
 142. The method of claim 141, wherein the methodis performed one or more times per week.
 143. The method of claim 142,wherein the method is performed one or more times per day.
 144. Themethod of claim 143, wherein the method is performed one or more timesper hour.
 145. The method of claim 1, wherein the method is performed atleast once, at least twice, at least three times, at least five times,or at least ten times.
 146. The method of claim 1, wherein the methodfurther comprises administering a treatment comprising IAIP or atherapeutic agent to the subject.
 147. The method of claim 146, whereinthe subject has an IAIP concentration of 200 μg/mL or lower.
 148. Themethod of claim 146, wherein the sample from the subject has an elevatedlevel of IAIP-IAIP ligand complex relative to a reference sample. 149.The method of claim 146, wherein the subject has or is at risk ofdeveloping an inflammatory disease or condition or an infection. 150.The method of claim 146, wherein the method comprises administering IAIPand a therapeutic agent to the subject.
 151. The method of claim 146 or150, wherein the therapeutic agent is selected from the group consistingof an antibiotic agent, an antiviral agent, an antifungal agent, anantiparasitic agent, an anti-inflammatory agent, an anti-cancer agent,an anti-coagulant, an immunomodulatory agent, a bronchodilator agent, acomplement inhibitor, a vasopressor, a sedative, or mechanicalventilation.
 152. The method of claim 1, wherein the subject has beenill for at least one day.
 153. The method of claim 152, wherein thesubject has been ill for at least one week.
 154. The method of claim153, wherein the subject has been ill for at least one month.
 155. Themethod of claim 154, wherein the subject has been ill for at least oneyear.
 156. The method of claim 1, wherein the method is for: c)evaluating the health status of the subject; d) monitoring the healthstatus of the subject; c) diagnosing the subject as having or being atrisk for an inflammatory disease or condition or an infection; d)evaluating efficacy of a treatment administered to the subject; or e)evaluating disease severity in the subject.
 157. The method of claim156, wherein the method further comprises comparing the amount of IAIPand/or an IAIP-IAIP ligand complex detected in the sample to the amountof IAIP and/or an IAIP-IAIP ligand complex found in a sample from anormal subject or to a cutoff value.
 158. The method of claim 157,wherein an amount of IAIP in the sample that is lower than an amount ofIAIP in the sample from the normal subject or relative to the cutoffvalue indicates that the subject has or is at risk of developing aninflammatory disease or condition or an infection.
 159. The method ofclaim 157, wherein an amount of an IAIP-IAIP ligand complex in thesample that is greater than an amount of IAIP-IAIP ligand complex in thesample from the normal subject or relative to the cutoff value indicatesthat the subject has or is at risk of developing an inflammatory diseaseor condition or an infection.
 160. The method of claim 157, wherein theamount of IAIP in the sample from the normal subject, or the cutoffvalue, is >250 μg/mL.
 161. The method of claim 160, wherein the amountof IAIP in the sample from the normal subject is about 260 to about 540μg/mL.
 162. The method of claim 1, wherein a determination that thesubject has an IAIP concentration of 250 μg/mL or less indicates thatthe subject has or is at high risk of developing an inflammatory diseaseor condition or an infection or is diagnosed as having an increased riskof morbidity and/or mortality.
 163. The method of claim 157, wherein thesubject has an IAIP concentration of 200 to 300 μg/mL and wherein themethod is performed at least once a year.
 164. The method of claim 163,wherein the method is performed at least twice a year.
 165. The methodof claim 163, wherein the method is performed at least once a month.166. The method of claim 163, wherein the method is performed at leastonce a week.
 167. The method of claim 163, wherein the method isperformed at least once a day.
 168. The method of claim 163, wherein themethod is performed at least once an hour.
 169. The method of claim 1,wherein the subject previously had an inflammatory disease or conditionor an infection.
 170. The method of claim 146, wherein the method isperformed prior to the treatment and one or more times during the courseof the treatment.
 171. The method of claim 146, wherein the method isperformed after initiation of the treatment and/or after conclusion ofthe treatment.
 172. The method of claim 146 wherein the treatment isdetermined to be effective if the concentration of IAIP increases in thesubject relative to a prior measurement of IAIP in the subject and/or ifthe concentration of an IAIP-IAIP ligand complex decreases in thesubject relative to a prior measurement of an IAIP-IAIP ligand complexin the subject.
 173. The method of claim 146, wherein the treatment isdetermined to be ineffective if the concentration of IAIP decreases orremains constant in the subject relative to a prior measurement of IAIPin the subject and/or if the concentration of an IAIP-IAIP ligandcomplex increases or remains constant in the subject relative to a priormeasurement of an IAIP-IAIP ligand complex in the subject.
 174. Themethod of claim 173, wherein the method further comprises modifying orchanging the treatment.
 175. A method of treating a subject that has oris at risk of developing an inflammatory disease or infection, whereinthe subject has been determined to be in need of treatment according tothe method of claim 1, comprising administering to the subject atherapeutically effective amount of IAIP and/or a therapeutic agentselected from the group consisting of an antibiotic agent, an antiviralagent, an antifungal agent, an antiparasitic agent, an anti-inflammatoryagent, an anti-cancer agent, an anti-coagulant, an immunomodulatoryagent, a bronchodilator agent, a complement inhibitor, a vasopressor, asedative, or mechanical ventilation.
 176. The method of claim 175,wherein the inflammatory disease or condition is selected from the groupconsisting of sepsis, septic shock, sterile sepsis, trauma, injury,stroke, acute inflammatory disease, SIRS, acute lung injury, ARDS,pneumonia, necrotizing enterocolitis, acute pancreatitis, renal disease,acute kidney injury, liver injury, acute circulatory failure,surgery-induced inflammation, abscess-induced inflammation, multiplesclerosis, preeclampsia, cancer, cancer metastasis, tumor invasion,peripheral artery disease, type 1 or type 2 diabetes, atheroscleroticcardiovascular disease, intermittent claudication, critical limbischemic disease, myocardial infarction, carotid occlusion, umbilicalcord occlusion, low birth-weight, premature birth, pulmonaryinsufficiency, peripheral neuropathy, hypoxic ischemia, bacterialinfection, wounds, burns, lacerations, contusions, bone fractures,surgical procedures, tissue ischemia, rheumatoid arthritis, meningitis,inflammatory bowel disease, chronic obstructive pulmonary disease,rhinitis, preterm labor, or an infectious disease.
 177. The method ofclaim 175, wherein the infection is caused by a gram negative bacteria,such as Neisseria species including Neisseria gonorrhoeae and Neisseriameningitidis, Branhamella species including Branhamella catarrhalis,Escherichia species including Escherichia coli, Enterobacter species,Proteus species including Proteus mirabilis, Pseudomonas speciesincluding Pseudomonas aeruginosa, Pseudomonas mallei, and Pseudomonaspseudomallei, Klebsiella species including Klebsiella pneumoniae,Salmonella species, Shigella species, Serratia species, Acinetobacterspecies; Haemophilus species including Haemophilus influenzae andHaemophilus ducreyi, Brucella species, Yersinia species includingYersinia pestis and Yersinia enterocolitica, Francisella speciesincluding Francisella tularensis, Pasturella species includingPasteurella multocida, Vibrio cholerae, Flavobacterium species,meningosepticum, Campylobacter species including Campylobacter jejuni,Bacteroides species (oral, pharyngeal) including Bacteroides fragilis,Fusobacterium species including Fusobacterium nucleatum,Calymmatobacterium granulomatis, Streptobacillus species includingStreptobacillus moniliformis, and Legionella species includingLegionella pneumophila.
 178. The kit of claim 81, wherein the detectionagent is labeled.
 179. The kit of claim 81, wherein the IAIP bindingagent is an IAIP-specific antibody or an IAIP ligand.
 180. The kit ofclaim 81, wherein the kit further comprises an IAIP ligand bindingagent.
 181. The kit of claim 180, wherein the IAIP ligand binding agentis an antibody that binds to an IAIP ligand.
 182. The kit of claim 81,wherein the IAIP detection agent is an IAIP-specific antibody or an IAIPligand.
 183. The kit of claim 81, wherein the kit further comprises anIAIP ligand detection agent.
 184. The kit of claim 183, wherein the IAIPligand detection agent is an antibody that binds specifically to an IAIPligand.
 185. The kit of claim 179 or 182, wherein the IAIP-specificantibody is a monoclonal antibody.
 186. The kit of claim 185, whereinthe monoclonal antibody is MAb 69.26 or MAb 69.31.
 187. The kit of claim82, wherein the support is a plate, a resin, a container, a membrane, abiochip, a particle, a nanoparticle, a test strip, or a bead.
 188. Thekit of claim 81, wherein the label is an enzyme, an enzyme substrate,biotin, a particle, a fluorescent dye, a luminescent compound, or aradiolabel.
 189. The kit of any one of claims 179-184, wherein the IAIPligand is selected from the group consisting of endotoxin (LPS),heparin, a histone, hyaluronic acid, laminin, tenascin C, aggrecan,vitronectin, fibronectin, von Willebrand Factor, pentraxin-3 (PTX3),TNF-stimulated gene-6 (TSG-6), factor IX, a complement component, factorXIIIa, and tissue transglutaminase.